In order to colonize the human gastrointestinal tract and exert their beneficial effects, bifidobacteria must effectively cope with the toxic bile salts in the intestine, but the molecular mechanism underlying bile tolerance is poorly understood. In this study, heterologous expression of a MarR family transcriptional regulator BmrR significantly reduced ox-bile resistance of Lactococcus lactis NZ9000, suggesting that it might play a role in bile stress response. In silico analysis combined with RT-PCR assay demonstrated that bmrR was co-transcribed with bmrA and bmrB, which encoded multidrug resistance (MDR) ABC transporters. Promoter prediction and EMSA assay revealed that BmrR could autoregulate the bmrRAB operon by binding to bmr box (ATTGTTG-6nt-CAACAAT) in the promoter region. Moreover, heterologous expression of bmrA and bmrB in L. lactis showed 20.77-fold higher tolerance to 0.10% ox-bile compared to wild type strain. In addition, ox-bile could disrupt the DNA binding activity of BmrR as a ligand. Taken together, our findings indicate that bmrRAB operon is autoregulated by transcriptional regulator BmrR and ox-bile serves as an inducer to activate the bile efflux transporter BmrAB in response to bile stress in B. longum BBMN68. Importance: Bifidobacteria are natural inhabitants of the human intestinal tract. Some bifidobacterial strains are used as probiotics in fermented dairy production because of their health-promoting effects. Following consumption, bifidobacteria finally colonize the lower intestinal tract where the concentration of bile salts remains nearly 0.05% to 2.0%. Bile salts as detergent-like antimicrobial compounds can cause disruption of the cellular membrane, protein misfolding and DNA damage. Therefore, tolerance to physiological bile stress is indeed essential for bifidobacteria to survive and exert the probiotic effects in gastrointestinal tract. In B. longum BBMN68, the MarR-type regulator BmrR was involved in bile stress response by auto-regulating bmrRAB operon and ox-bile as an inducer could increase the expression of BmrAB transporter to enhance the bile tolerance of BBMN68.This is the first report about functional analysis of bmrRAB operon in bile stress response, which will provide new insight into bile tolerance mechanisms in Bifidobacterium and other bacteria.