The aim of this study was to investigate the in vivo transfer of aflatoxin B1 (AFB1) to Assaf ewes' milk (aflatoxin M1, AFM1) and its effect on animal performance and health. Thirty Assaf ewes were allocated to three groups (C, L, H), and received a different individual daily dose of AFB1 (0, 40 and 80 μg) for 13 days. Milk (days 1, 2, 3, 4, 7, 14, 16 and 18) and blood (days 1, 7, 14 and 18) samples were collected. Milk yield, composition (except protein) and somatic cell counts (SCC) were not affected by AFB1 intake (p > 0.05). Haemoglobin concentration increased (p < 0.05) and haematocrit and alanine aminotransferase levels tended to increase (p < 0.10) in group H on day 14. AFM1 excretion was highly variable and detected in L and H animals from days 1 to 16 (3 days increase, 10 days steady-state, 3 days clearance). Carry-over rate (0.23%) was significantly higher in L (0.22-0.34%) than in H (0.16-0.19%) animals (p < 0.05). AFB1 daily doses of 40 to 80 µg do not impair milk yield; however, it may start affecting animals' health. Milk AFM1 depends mainly on the AFB1 intake whereas carryover rate is positively influenced by the level of milk production.