Background Mycobacterium tuberculosis differs from most pathogens in its ability to multiply inside monocytes and to persist during long periods of time within granuloma in a status of latency. A class of proteins called mycobacterial histone-like proteins has been associated with regulation of replication and latency, but their precise role in the infection process has yet to be uncovered. Our study aimed at defining the impact of the histone-like protein MDP1 from M. bovis BCG (mycobacterial DNA-binding protein 1, corresponding to Rv2986c from M. tuberculosis) on early steps of infection. Results Previously, a BCG (Bacillus Calmette Guérin) strain had been generated by antisense-technique exhibiting reduced MDP1 expression. This strain was now used to analyse the impact of reduced amount of MDP1 on the interaction with human blood monocytes, macrophage lines and PBMC (peripheral blood mononuclear cells). MDP1 was revealed to be required for growth at acidic pH and for intracellular replication in human blood monocytes. Down-regulation of MDP1 resulted in reduced secretion of the cytokine IL-1β by infected human PBMC. In addition, a reduction of MDP1 expression had a major impact on the formation of fused multi-nucleated macrophages. In monocyte preparations from human blood as well as in human and mouse macrophage cell lines, both the percentage of multi-nucleated cells and the number of nuclei per cell were much enhanced when the monocytes were infected with BCG expressing less MDP1. Conclusion MDP1 from M. bovis BCG affects the growth at acidic pH and the intracellular replication in human monocytes. It furthermore affects cytokine secretion by host cells, and the formation of fused multi-nucleated macrophages. Our results suggest an important role of MDP1 in persistent infection.