Aim: To study the interference of organophosphate pesticide chlorpyrifos (CPF) in gonad function at the steroidal and gene levels during prespawning adult and fry stage respectively of the freshwater stinging catfish, Heteropneustes fossilis. Methods: The matured adult fish of early prespawning stage were exposed to different combinations of CPF (0.174 μM, 10-1th of 96 h LC50), gonadotropin (hCG; 100 IU.fish-1) and natural antioxidants (curcumin; 10 mg.L-1 and ascorbic acid; 5 mg.L-1) for 24 h to measure vitellogenin and ovarian steroid. Twenty days post-hatch, fish fry were exposed to CPF for 96 h to understand the influence on NR5A1 gene expression pattern. Results: The CPF increased vitellogenin level at liver, ovary and serum. Among steroidal cascade CPF induced highest estradiol followed by a significant increase in progesterone and 17α, 20β-dihydroxyprogesterone as compared to control fish. The gonadotropin further increased CPF induced effects on vitellogenin and ovarian steroidal level. Though, antioxidants co-incubation with CPF did not produce any significant change on either vitellogenin or in ovarian steroids content. CPF triggers NR5A1 gene expression in a fry that indicates a role in early gonad differentiation and development. Conclusion: The present study clearly indicates the major influence of CPF in gonadal physiology of both adult and fry stages of freshwater catfish, Heteropneustes fossilis.