Enterococcus faecalis is closely associated with refractory apical periodontitis, manifesting periapical lesions and alveolar bone loss. Macrophages playing an important role in the induction of inflammation can differentiate into bone-resorbing osteoclasts. In the present study, we investigated the effect of E. faecalis on the differentiation and function of macrophages as osteoclast precursors.Bone marrow–derived macrophages (BMMs) were differentiated into osteoclasts with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand in the presence or absence of heat-killed E. faecalis (HKEF). Tartrate-resistant acid phosphatase–positive multinucleated giant cells were analyzed to determine osteoclast differentiation. Western blotting was performed to examine the expression of c-Fos and NFATc1 transcription factors. Phagocytic capacity was analyzed by measuring uptake of carboxyfluorescein succinimidyl ester–labeled E. faecalis. Secretion of tumor necrosis factor-α, interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1 was determined by enzyme-linked immunosorbent assay.Differentiation of BMMs into osteoclasts was attenuated in the presence of HKEF, and expression of c-Fos and NFATc1 was inhibited. HKEF exposure also prevented a reduction in the phagocytic capacity of BMMs after differentiation into osteoclasts. Concomitantly, HKEF induced the expression of chemokines monocyte chemotactic protein-1 and keratinocyte-derived chemokine and proinflammatory cytokines tumor necrosis factor-α and interleukin-6.E. faecalis attenuated macrophages from differentiating into osteoclasts, allowing them to keep their ability to phagocytose and kill pathogens and to induce proinflammatory cytokine and chemokine secretion.