The impact of heavy metal levels (Cd, Cu, and Zn) on the reproductive parameters in natural populations of the Mediterranean killifish Aphanius fasciatus was monitored in four sites of the Tunisian coast. Our study covered a period of the reproductive cycle, from April to November 2009. The significantly higher levels of Cd, Cu, and Zn in the liver and gonads for both sexes (p<0.05) were observed in the most polluted site (Sfax coast). During the reproductive cycle of A. fasciatus, an unbalanced sex ratio in favor to females was observed for all the studied populations. The progression of the hepato- (HSI), gonadosomatic (GSI), and K-factor revealed a spatio-temporal variation between populations during the sampling period. Estradiol-17β amounts in the gonad tissues of females captured from the studied sites showed a significant difference, and the highest concentrations were noticed in the control site (Luza). Taking together, our data provide the first evidence of a physiologically stress and a disturbance of the reproductive status in natural populations of A. fasciatus.