The high prevalence of Mycobacterium tuberculosis (MTB) despite widely available Bacille Calmette-Guerin (BCG) vaccination may be associated with Mycobacterium avium (M. avium), which may influence the host response to MTB. In this study, we demonstrate that pretreatment of murine macrophages with low-dose Mycobacterium avium-derived lipids (MALs), but not Escherichia coli-derived lipids (ELs), attenuates the clearance of intracellular Mycobacterium bovis BCG (M. bovis BCG) and the production of TNF-α, IL-6, IL-12 and nitric oxide (NO) following stimulation with purified protein derivatives (PPD) or heat-inactivated M. bovis BCG in vitro. Furthermore, a significant decrease in NF-κB activity was observed in MALs-pretreated RAW264.7 cells that were co-transfected with pSV-β-galactosidase and pGL4.32[luc2P/NF-κB-RE/Hygro] prior to stimulation with PPD or heat-inactivated M. bovis BCG. In contrast, IRAK-M, an inhibitor of NF-κB activation, was increased in these cells. This observed hyporesponsiveness is not related to the expression of Toll-like receptor 2 (TLR2). In conclusion, pretreatment with low-dose MALs can induce hyporesponsiveness to MTB components in murine macrophages.