Abstract Proteoliposomes (PL) obtained from Mycobacterium smegmatis (Ms) were evaluated for their capacity to elicit cross-reactive responses against Mycobacterium tuberculosis (Mtb) antigens in BALB/c mice. Animals immunized with PL adjuvanted with alum (PL–AL) or Freund's Incomplete Adjuvant (PL–IFA) showed significant IgG responses against the PL as well as total Ms lipids. Both groups of animals also showed significant IgG responses against BCG, but only animals immunized with PL–AL produced significant IgG responses against soluble cell wall proteins (SCWP) or whole cell lysate (WCL) of Mtb. Significant DTH responses against WCL were observed in both groups of animals after 24h, but only PL–AL-immunized mice showed significant DTH responses after 48h and 72h. PL–Ms are capable of eliciting cross-reactive humoral and cellular responses against Mtb antigens and thus may be a potential vaccine strategy against tuberculosis.