Equine reproductive behavior is affected by many factors, some remaining poorly understood. This study tested the hypothesis that a period of captivity during the juvenile period and human-controlled reproduction may potentially be involved in the disruption of the development of incestuous mating avoidance behavior in sanctuary-reintroduced male Konik polski horses. Between 1986 and 2000, cases of incestuous behavior in harem stallions born and reared until weaning in the sanctuary were studied. Eight males lived in the sanctuary's feral herd for the rest of their lives (the non-captive group; nC). They gained their own harem of mares without human intervention (no human-controlled reproductive activity, nHC). Another five stallions were removed as weanlings, reared in captivity and then reintroduced as adults (captive, C). Three of these C stallions were used as in-hand breeding stallions, one as a "teaser" (human-controlled reproductive activity, HC) and one was not used for reproduction in captivity (nHC). Reproductive records for 46 mares, daughters of all 13 harem stallions, were scrutinized and cases of incestuous breeding were recorded by interrogation of foal parentage records. C stallions failed to expel more daughters than nC stallions (33% vs. 18%, P = 0.045), and mated with significantly more of them (28% vs. 11%, P = 0.025). Interestingly, HC stallions expelled fewer (60%) and successfully mated with more (33%) daughters that nHC stallions (84% expelled, P = 0.013, and 10% successful mating with daughters, P = 0.010). All HC stallions bred incestuously at least once. We propose that human intervention during a critical period of development of social and reproductive behavior in young stallions, by enforced separation from their natal herd and in-hand breeding, may contribute to their later aberrant behavior and disruption of inbreeding avoidance mechanisms in these stallions. The previous occurrence of human-controlled breeding may be one of the factors promoting incestuous behavior of stallions in natural conditions. The uninterrupted presence of stallions in their harems and herd member recognition may also play important roles in inbreeding avoidance in horses.