Abstract Background Understanding pathogenic mechanisms is imperative for developing novel treatment to the tuberculosis, an important public health burden worldwide. Recent studies demonstrated that host cholesterol levels have implications in the establishment of Mycobacterium tuberculosis (M. tuberculosis, Mtb) infection in host cells, in which the intracellular cholesterol-mediated ATP-binding cassette transporters (ABC-transporters) and cholesterol acyltransferase1 (ACAT1) exhibited abilities to regulate macrophage autophagy induced by Mycobacterium bovis bacillus Calmette–Guérin (BCG). Results The results showed that a down-regulated expression of the ABC-transporters and ACAT1 in primary bovine alveolar macrophages (AMs) and murine RAW264.7 cells in response to a BCG infection. The inhibited expression of ABC-transporters and ACAT1 was associated with the reduction of intracellular free cholesterol, which in turn induced autophagy in macrophages upon to the Mycobacterial infection. These results strongly suggest an involvement of ABC-transporters and ACAT1 in intracellular cholesterol-mediated autophagy in AMs in response to BCG infection. Conclusion This study thus provides an insight into into a mechanism by which the cholesterol metabolism regulated the autophagy in macrophages in response to mycobacterial infections.