Abstract: The present study was carried to observe the effect of aflatoxin on ovine sperm in vitro. Ram epididymal and ejaculatory sperm cells were added into media containing different concentrations of aflatoxin B1. When ram epididymal sperm were put in different concentrations of aflatoxin, one-hour post incubation in control group, 81.25% of sperm cells were alive of which, 82.88% were motile. Significantly (p< 0.05) higher than medium with 31.25 ppb and higher concentration. The lowest motility (15.93%) was observed in 62.5 ppb aflatoxin. Sperm viability did not significantly changed after 2nd and 3rd h incubation but significantly decreased in 4th and 5th h post incubation. However, motility was not altered in the 2nd, 3rd, 4th and 5th h post incubation. In ejaculatory sperm, one hour post-incubation, in control group, 83.24% of sperm were alive of which 93.98% were motile, significantly (p< 0.05) higher than motility in medium with 7.81 ppb and higher concentrations of aflatoxin but not different with 1.96 ppb aflatoxin. In 62.5 ppb aflatoxin, the lowest motility was observed (18.9%). Sperm motility patterns for both epididymal and ejaculatory sperm cells were different after incubation in different concentrations of aflatoxin. The results of the present experiment showed that aflatoxin could decrease sperm motility obtained from ejaculation or epididymis.