Objective To investigate the expression of inducible nitric oxide synthase ( iNOS) and the levels of nitric oxide (NO) in THP-1 and J774A. 1 cells during Leptospira interrogans (L. interrogans) infection for a better understanding of the mechanism of macrophages involved defense against L. interrogans strains in different hosts. Methods The human mononuclear macrophages (THP-1) and the murine mono-nuclear macrophages (J774A. 1) were infected with L. interrogans strain 56601. The expression of iNOS at mRNA and protein levels were determined by using real-time RT-PCR and flow cytometry analysis. The lev-els of NO were detected with Griess test. Results The expression of iNOS at mRNA level in J774A. 1 and THP-1 cells infected with L. interrogans strains for 2, 4, 12 and 24 hours were respectively 1. 37, 2. 82, 25. 76, 27. 47 times and 1. 59, 3. 98, 3. 89, 8. 81 times than that of cells without infection (P<0. 05). The expression rates of iNOS protein in J774A. 1 cells were increased from 34. 16% to 85. 85%, 93. 82%, 91. 77% and 93. 65% along with the increased time of infection time (P<0. 05). The expression rates of iNOS protein in THP-1 cells were up-regulated from 22. 08% to 72. 64%, 81. 33%, 80. 03% and 65. 72%after 2, 4, 12 and 24 hours of infection (P<0. 05), respectively. Results of the Griess test indicated that the levels of NO in J774A. 1 and THP-1 cells were respectively increased from 0. 1588 μmol/L to 0. 2208μmol/L, 0. 2668μmol/L, 0. 3808μmol/L, 0. 3828μmol/L and from 0. 0988μmol/L to 0. 2848μmol/L,0. 3228 μmol/L, 0. 2608μmol/L and 0. 3308μmol/L after infection with L. interrogans strains for 2, 4, 12 and 24 hours (P<0. 05). Conclusion The expression of iNOS and the levels of NO in J774A. 1 and THP-1 cells were significantly increased during L. interrogans infection. This study might help to explain the bactericidal mechanism of macrophages derived from different hosts against L. interrogans infection.