A large amount of estrogen is introduced into the feed to improve livestock growth, excessive residual estrogen would exist in the excrements. The accumulation of estrogen can cause the water and soil pollution around the farm, further inhibit growth and induce caner for organisms and human. Anaerobic digestion (AD) could effectively remove estrogen in livestock manure while the mechanism is still unclear. In this study, the mechanism of estrogen removal by AD was explored during AD process of pig manure. Estrone (E1), estradiol (E2) estriol (E3) and ethinylestradiol (EE2) were selected as research subjects. The removal rates of E1, E2, E3 and EE2 were 19.14, 28.62, 25.83 and 11.81%. Dissolved organic matters (DOM), especially humic acid, play an important role in reducing bioavailability of estrogen. Estrogen will be absorbed by DOM through structures such as aromatic ring and amides. Estrogen may also promote the growth of microorganisms which could degrade estrogen, such as Rhodococcus, Sphingomonas and Pseudomonas. The amount of these microorganisms and dissolved microbial metabolites did not change obviously. This study would give a new explanation for the removal of estrogen and can provide theoretical support for harmless treatment of pig manure.Graphical Abstract: