In human and mice, the ABC efflux transporter ABCG2 represents the main route for active drug transport into milk. In contrast, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix®, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by HPLC. Compared to the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as AUC). Moreover, we observed MNPSO2 excretion into milk of dairy cows with high milk to plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral to apical (B>A) MNPSO2 transport across polarized MDCKII-bABCG2 monolayers using LC-MS/MS analysis. The B>A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in Mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocylic lactone anthelmintic moxidectin caused a reduction in the MNPSO2 (B>A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk.