This investigation was intended to find out the estrogenic effect of chlortetracycline (CTC) on vitellogenin induction in adult male Japanese medaka (Oryzias latipes). Vitellogenin (Vtg) produced in male fish has been used to as one of a biomarker of endocrine disrupters. The positive control was 17β−estradiol (E2) that induced Vtg in male fish. As a result, male and female fish were exposed to 0.1, 1, 10 and 100 ppm of CTC. Western blot results showed approximately 205 kDa, that is similar to myosin at high molecular weight range Sigma maker. Vtg band was showed fainted to 10 and 100ppm for chlortetracycline. Vtg concentration of CTC was qunatified by total protein quantification and ELISA. Exposure of the male fish to CTC of 0.1, 1, 10 and 100 ppm produced Vtg concentrations of 0.24, 0.12, 7.61 and 40.02%o, respectively, that value was elevated than control male fish (0.14%o). CTC exerted as a Vtg inducer in male fish from 10 ppm, but it was a reducer in female fish from 0.1 ppm level. The results say that vitellogenin induction patterns alter in male medaka treated with CTC, and that CTC may caused endocrine disruption in fish.