Lipopolysaccharide (LPS) induces apoptosis in murine macrophages through the autocrine secretion of tumor necrosis factor (TNF)-α and nitric oxide (NO). LPS-induced inflammation in murine macrophages is associated with hydrogen sulfide (H₂S) production. In this present study, we reported the novel role of H₂S in LPS-induced apoptosis and its underlying molecular mechanism specifically at late phases in murine macrophage cells. Stimulation of RAW 264.7 macrophages with LPS resulted in a time- and dose-dependent induction of apoptosis. We observed that the LPS-induced early apoptosis (associated with TNF-α secretion) in macrophages was not inhibited in the presence of H₂S inhibitor (DL-propargylglycine), whereas early apoptosis was absent in the presence of TNF receptor antibody. Interestingly, LPS-induced late apoptosis paralleled with H₂S production was reduced in the presence of H₂S inhibitor but not with TNF receptor antibody. The late apoptotic events mediated by H₂S and not the TNF-α induced early apoptosis correlated significantly with the induction of p53 and Bax expression in LPS-induced macrophages. Thus, it is possible that RAW 264.7 murine macrophages treated with LPS mediated early apoptosis through TNF-α and the late apoptotic events through the production of H₂S.