Insect pests are one of the major factors affecting crop production and a variety of chemical insecticides are being used to control pests, but due to their hazardous nature there is a need of alternative options in the form of biological control. The biological insecticides obtained from Bacillus thuringiensis (Bt) have been introduced almost two decades ago but resistance against Bt in field population of insects has been reported. Recently a new class of biological toxin gene, ω-HXTX-Hv1a (Hvt) from the Australian funnel web spider Hadronyche versuta has been introduced into cotton. This study evaluated the possible adverse effects of Hvt gene containing transgenic cotton through risk assessment studies. Sub-chronic feeding study was conducted in albino rabbits to evaluate the potential health effects of long-term consumption of a rodent diet containing Hvt cottonseed compared with a diet containing cottonseed from its near-isogenic control. Acute and sub-chronic studies in mice and rabbits showed that NOAEL was greater than 5000 mg per kg body weight of the test animals. No toxicologically significant differences were observed in nutritional performance variables, clinical hematology and chemistry between mice and rabbits fed with the Hvt cottonseed or purified protein from transformed Pichia and E. coli cells when compared to control treatment groups. Allergenicity assessment through primary skin irritation , mucous membrane irritation and primary ocular irritation tests showed that Hvt gene encoded toxin in the cotton or Pichia cells is safe ansd poses no risk as an allergen when applied to intact skin or mucous membrane. Agraonomic and yield parametrs of the Hvt cotton showed non-significant differences except their ability to resist the lepidopteran insect attack. Compositional anlysis of the Hvt cotton also indicated similar levels of the proteins, aminoacids, gossypol and carbohydrate content of the Hvt cotton is substantially equivalent to non-transgenic cotton. It was observed that the transgenic cotton carrying functional Hvt gene had non-significant effect on reproductive characteristics (pollen morphology, longevity and fertility), plant germination and soil microflora as compared to non-Hvt cotton. Moreover, root, stem and leaf extracts of cotton expressing Hvt gene showed non-significant allelopathic effect on the germination of tobacco seeds. Similarly, pests scouting of Hvt and non-Hvt cotton stands indicate that there was non-significant variation in the number of the insect species and their population size between Hvt and non-Hvt cotton, but the population density of H. armigera larvae was significantly low on Hvt cotton. These findings conclude that the transgenic cotton expressing Hvt gene did not poses any considerable risks or harm to the plant morphology, physiology and its surrounding environment and might be useful candidate gene against Lepidopteron pests.