Pentachlorophenol (PCP) is the most toxic contaminant of chlorophenols (CPs). Due to improper disposal, PCP has become an environmental pollutant and is now considered to be ubiquitous. Previous studies about the influences of PCP on reproductive function were mostly focused on experiments in vivo. The aim of our present study was to estimate the toxic effects of PCP on cultured Sertoli cells from Sprague-Dawley rats. The viability of Sertoli cells was detected and morphological examination was performed followed by flow cytometric assay to evaluate its toxic effects. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that the toxic effects of PCP on cultured Sertoli cells were dose- and time-dependent. By flow cytometric assay, it was found that the number of dead Sertoli cells increased with the increase of exposed PCP levels. The results indicated that PCP had direct and dose-dependent cytotoxic effects on Sertoli cells in vitro.