Cotton fibers serve as the chief raw material for the textile industry. Cost of production and requirement of high-quality products is the main challenge encountered by the textile industry in Pakistan. Fiber quality parameters that have a major impact on the textile processing include fiber length, fiber strength and fiber fineness. Efforts to improve cotton fiber quality have been underway since many years. Out of all the approaches used so far, genetic improvement in cotton through Agrobacterium mediated transformation has proved to be the most feasible and productive technique in enhancing the fiber quality of various cotton cultivars. Cotton fibers are chiefly composed of cellulose therefore, any change in the cellulose synthesis within the fiber can affect the fiber quality to a great extent. Due to this fact, this study was aimed to explore the expression of bacterial cellulose synthase (Bcs) genes, namely acsA and acsB (GenBank ID: X54676.1), under the regulation of fiber-specific promoter (GhSCFP) in a local cotton variety (CEMB-00) of Gossypium hirsutum. In silico studies were conducted to understand the structures of bacterial and cotton fiber cellulose synthase enzymes and to find compatibility and similarities among them. A number of domains were found to be highly conserved among the bacterial and cotton fiber cellulose synthases. These conserved domains were observed to be functionally essential for the process of cellulose synthesis in the bacterial cells of Gluconacetobacter xylinus as well as the cotton fiber. The 6586 bp fragment of Bcs gene cassette was isolated from the synthesized pUC57-Bcs construct through restriction digestion analysis and cloned into pCAMBIA 1301 vector under the regulation of the fiber-specific promoter. The recombinant plant expression vector, pCAMBIA-Bcs, was introduced into the local non-transgenic cotton variety of Gossypium hirsutum via shoot apex method of Agrobacterium-mediated transformation which resulted in the transformation efficiency of 1.27%. Four out of ten putative transgenic plants, which were PCR positive for both acsA and acsB genes, were selected on the basis of improved fiber quality and better growth potential to be further analyzed through the molecular and biochemical analysis in the advanced generations. Quantitative expression of the acsA and acsB genes in the T1 generation transgenic plant lines was many folds higher at 35 DPA (during the secondary cell wall synthesis phase of fiber) as compared to expression at 10 DPA (during the elongation phase of fiber). Fiber analysis of the transgenic cotton plant lines showed up to 17.52% increase in fiber length, up to 26.45% increase in fiber strength and up to 22.45% increase in the cellulose contents as compared to the control (non-transgenic) plant line. SEM analysis showed that the transgenic fibers had smoother surfaces with more number of twists as compared to the control fibers. Improvement in some of the morphological traits was also observed in the transgenic plant lines showing an increase of 28.06-32.21% in plant height, 28-40% increase in number of bolls per plant and 31.21-44.57% increase in yield per plant. Furthermore, karyotyping results obtained through FISH analysis revealed the presence of single copy number of the transgenes on the chromosome no. 11 in the transgenic plant line S-00-16 and on chromosome no. 13 in the transgenic plant line S-00-11, during the metaphase. In light of the results obtained, it can be concluded that the transformation of the acsA and acsB genes in cotton fiber resulted in enhanced fiber quality on the basis of analyses performed on a small number of T1 plants. In order to meet the high standards of the cotton fiber quality for the national textile industry, replicated experimentation is required on a larger scale before firm conclusions can be made on parameters such as yield and fiber processing quality.