Leptospira interrogans is the major causative agent of leptospirosis, an emerging, globally spreading zoonotic infectious disease. The pathogen induces macrophage apoptosis, but the molecular basis and mechanism remain unknown. In the present study, we found that L. interrogans caused apoptosis of phagocytosis-inhibited macrophages, and the product of the L. interrogans LB047 gene (Lep-OMP047) was the unique protein captured by mouse and human Fas proteins. The recombinant expressed Lep-OMP047 (rLep-OMP047) strongly bound mouse and human Fas proteins with equilibrium association constant (KD) values of 5.20 × 10-6 to 2.84 × 10-9 M according to surface plasmon resonance measurement and isothermal titration calorimetry. Flow-cytometric examination showed that 5 μg rLep-OMP047 or 1 μg lipopolysaccharide of L. interrogans (Lep-LPS) caused 43.70% or 21.90% early apoptosis in mouse J774A.1 macrophages and 28.41% or 15.80% for PMA-differentiated human THP-1 macrophages, respectively, but the apoptosis was blocked by Fas-antagonizing IgGs, Fas siRNAs, and caspase-8/-3 inhibitors. Moreover, Lep-OMP047 was significantly upregulated during infection of macrophages. Lep-LPS promoted the expression and cytomembrane translocation of Fas and FasL in macrophages. The JNK and p38 MAPK but not ERK signaling pathways, as well as the transcription factors c-Jun and ATF2 but not CHOP, mediated Lep-LPS-induced Fas/FasL expression and translocation. TLR2 but not TLR4 mediated Lep-LPS-induced JNK/p38 MAPK activation. Therefore, we demonstrated that a novel Fas-binding OMP and LPS of L. interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway.