The effect of Cucurbita pepo extract and 17 b-estradiol (E2) on HPG axis was investigated in male Three spot gourami. The purpose of this research was evaluated effect of Cucurbita pepo on sex steroids level and measurement of blood calcium and also changes of liver tissue. Therefore, 100specimen of immature male fish with mean weight (2.5±0.5g) were evaluated in 10 treatments. The fishes in different groups were treated with Cucurbita pepo extract and the E2 separately. (10, 20, 30,50mg/Kg body weight).There was two groups as controls. Control I received 20mL methanol and control II was intact. After 20 days experimental, fishes were dissected. To measure the steroid hormones and calcium ion, tissue fluids were separated and the level of estradiol, testosterone and calcium ion analyzed. Results showed that level of sex steroid and Ca+2, for Cucurbita pepo extract in treatments I and II were 1.1, 0.83, 19.73, for treatment III were 1.34, 1.4, 20.65, for treatment IV were 1.8, 1.68, 22.86 and level of sex steroid and Ca+2, for (E2) in treatment V were 0.88, 2.55, 24.43 and VI treatment was 0.91, 3.54, 27.15 and for treatment VII were 0.97,4.41,30.80, for treatment VIII were 1.08, 6.45, 35.32 in comparison with control group (0.78, 0.52, 16.46) that were significant (P<0.05). These results showed that treatments IV and VIII were more affected by level steroids and Ca +2 ions in male Three spot gourami. The overall conclusion is that extract of Cucurbita pepo could possibly induce sexual maturity in male Three spot gourami.