[Objective] To investigate the effects of glutathione (GSH) on proliferation and biochemical function of primary hepatocyte in hybrid tilapia (Oreochromis niloticus×O. aureus).[Method] Tilapia primary hepatocytes were cultured in cell culture medium with 0 or 10% fetal calf serum for 48 h. GSH was then added into the two kinds of cell culture medium to the final concentration of 0, 30, 100, 300 or 900 mg·L^–1 respectively. Each concentration group had six replicates. The proliferation status of the primary hepatocyte, contents of insulin-like growth factor 1 (IGF-1), albumin and H₂O₂, as well as glutamate-oxaloacetate transaminase (GOT) and γ-glutamyl transpeptidase (γ-GT) activities of tilapia were detected after cultured for 24, 48 and 72 h, respectively.[Result] GSH significantly increased the proliferation of primary hepatocyte,the contents of IGF-1 and albumin,and γ-GT activity in supernatant,while reduced H₂O₂ content and GOT activity(P<0.05). Such effects of GSH were more evident in the group without fetal calf serum compared to the group with 10% fetal calf serum.[Conclusion] GSH could enhance the proliferation of primary hepatocyte and has the function of protecting liver in hybrid tilapia.