Brucella is an intracellular facultative bacterium able to survive and multiply in professional and non-professional phagocytes. However, its adhesion and invasion mechanisms have not been elucidated yet. In this work, we assess the interruption of a BMEI0216 gene of Brucella melitensis, by using HeLa epithelial cells and murine macrophages for invasion and replication assays. The mutation did not affect survival or multiplication within macrophages. Likewise, invasion assays with HeLa cells revealed no differences at 30 and 45 min, whereas, at 1 and 2h, the infection ability of the mutant was drastically reduced. These results suggest that the BMEI0216 gene is required for B. melitensis internalization.