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Water pollution of the Yangtze River in China became one of challenges that the government is facing today. Increasing numbers of petrochemical plants were built along the river in past decades, and numbers of organic chemicals were discharged into the river. Our goal was to establish in vitro system on rat sertoli cells, spermatogenic cells and leydig cells to investigate the reproductive toxicity potential induced by organic extracts from petrochemical effluents. Our results showed that the organic extract depressed the viability (p < 0.01) and destroyed the plasma membrane integrity of sertoli cells and spermatogenic cells to a certain degree. Accordingly, proportion of early apoptotic sertoli cells and late apoptotic spermatogenic cells increased significantly. Although significant morphological changes were not detected for leydig cells, the extract was observed to inhibit their testosterone production (p < 0.01). Sertoli cells and spermatogenic cells appeared to be more sensitive and maybe the main targets of the key toxins. These results suggested that the in vitro system on rat testicular cells may be useful to predicate reproductive toxicity potential of organic extracts from petrochemical effluents. More attention should be paid to the petrochemical effluents, because long-term accumulation of these organic compounds in organisms may cause spermatogenesis malfunction and testosterone reduction.