Sperm is the core of male fertility, which has to travel up to the fallopian tube for successful fertilization. Sperm motility depends on the electron transport chain producing ATPs in its mitochondria, which is a direct expression of the mitochondrial DNA (mtDNA) quality. Sperm motility is major determinant of fertility. It is already believed that mtDNA mutations are linked with infertility but the results are contradictory and previous researches are based on limited number of semen samples. Previous studies indicated a vacuum for more comprehensive study of sperm mtDNA from multiple aspects with sufficient number of carefully selected subjects to find more concrete findings. This case control study was designed on these hard facts to find association of sperm mtDNA deletions with fertility. We hypothesized that sperm mtDNA deletions have significantly associations with human male infertility. We collected 355 human semen samples (following WHO protocols), 74 samples normal controls (produced at least one child) and 281 infertile patients. Infertile samples were further classified into five groups, asthenozoospermia (As), oligozoospermia (Oz), oligotetrozoospermia (Ot), oligonecrozoospermia (On) and oligoasthenoteratozoospermia (OAT syndrome). Infertile nfertile nfertile nfertile individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. individuals were cases of idiopathic infertility. We focused on the most coherent core part of sperm mtDNA, the COXIII subunit. DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted DNA from semen samples was extracted by modified organic protocol and the DNA quantification was carried out spectrophotometrically at 260nm/280nm. The data for control and infertile patients was compiled and analyzed with IBM SPSS version 22 (SPSS, Chicago, IL, USA) Chi Square test was applied and P value less than (P=0.05) was considered as significant. The famous deletion (9480del15bp) was analyzed with COXIIIA and COXIIIB pair of primers and data analysis revealed highly significant association between infertile and deletions (P=0.001). In second set of experiments a bigger segment (50bp upstream to 9480del15) was explored with COXIIIC and COXIIIB primers of which the frequency of mutations was significantly higher in OAT samples (P=0.038). In third experiment multiple deletions were amplified simultaneously in larger segment by long PCR with MT1A and MT3 primers data analysis revealed highly significant association (P=0.001). Multiple mtDNA mutation had direct relation with poor semen quality and maximum incidence of mutations was identified in OAT syndrome. Sequencing analysis of COXIII indicated a polymorphism at position 9505, transition of C>G in all sample analyzed. Another transverse of codon at position 9526 marked by the change of C>A was discovered. This study demonstrated that sperm mtDNA deletions have significant roles in the diminution of fertility by deteriorating various sperm motility parameters. All four set of experiment revealed similar pattern of deletions associated to male infertility, highest incidence found in OAT group. The results are conclusive of “the male infertility is significantly associated to sperm mtDNA deletions.” We estimated that simple semen analysis parameters might be predictive of sperm mtDNA quality.