Abstract [Objective] This study aimed to investigate the effect of zearalenone （ZEN） on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose （LD~o） of ZEN with tetra- zolium-based colorimetric assay （MTT assay）. Comet assay was carried out to de- tect the DNA damage of porcine leydig cells exposed to at 0 （negative group）, 1, 5, 10, 20, 40 tJmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%. 52.00% and 64.67% under 0, 1, 5, 10 and 20 ~mo~/L o~ ZEN, respectively; the differences between the percentages of celt tail in various experimental groups had extremely significant statistical significance compared with the negative group （P〈0.01）, showing a significant dose-effect relationship; Tail length in various groups was 57.60_＋4.78, 57.75_＋6.25, 78.97_＋5.83, 100.50~6.94 and 146.83_＋12.31 ~m, re- spectively; Tail DNA % in various groups was 21.29_＋2.25%, 22.24_＋2.43%, 31.21_＋ 6.27%, 37.45_＋4.33% and 60.68_＋9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 ~mo~/L showed significant dif- ferences （P〈0.05） compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.