Betatrophin is regarded as a liver-produced hormone induced by insulin resistance (IR). However, it remains largely unknown how IR regulates betatrophin expression. To study whether IR could regulate betatrophin expression and the corresponding molecular mechanisms, betatrophin levels were examined in 6 in vitro IR models which were established using human hepatocytes L02 with different agents, including tumor necrosis factor-alpha, interleukin-1 beta, dexamethasone, palmitate, high glucose and insulin and betatrophin levels were elevated only in the insulin group. These results suggest that it is insulin, not IR that promotes betatrophin expression. In the meantime, PI3K/Akt pathway was activated by insulin and suppressed by above agents that caused IR. Insulin-upregulated betatrophin expression was suppressed by PI3K/Akt inhibitors and IR, suggesting that insulin upregulates and IR decreases betatrophin production through PI3K/Akt pathway. Consistently, the treatment of insulin in mice dose-dependently upregulated betatrophin levels, and the administration of metformin in IR mice also stimulated betatrophin production since published study showed metformin improved PI3K/Akt pathway and IR. In humans, compared with those without insulin treatment, serum betatrophin levels were increased in type 2 diabetic patients with insulin treatment. In conclusion, insulin stimulates betatrophin secretion through PI3K/Akt pathway and IR may play an opposite role.