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High mobility group box 1 protein (HMGB1), a nuclear protein, is a critical cytokine that mediates the response to infection, injury, and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB1 from supernatants of cells, following induction with Mycobacterium bovis BCG. Densitometric analysis revealed higher concentrations of HMGB1 in cell supernatants stimulated with BCG than in the supernatants of the control, nonstimulated cells. Further quantitation of the secreted HMGB1 was performed by ELISA. The BCG strain resulted in a higher amount of secreted HMGB1 (450 ± 44 ng/mL) than that of LPS (84 ± 12 ng/mL) or Staphylococcus aureus (150 ± 14 ng/mL). BCG and Phorbol −12-myristate −13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 ± 125 ng/mL). The translocation of the HMGB1 towards the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations. Conclusion: Our pilot experiments draw attention to the HMGB1 inducing ability of Mycobacterium bovis. Assessment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.