Inducible nitric oxide synthase (iNOS) contributes critically to inflammation and host defense. While normally undetectable, iNOS expression is induced by endotoxins and cytokines via discrete signaling pathways. Lipopolysaccharide (LPS) triggers iNOS gene transactivation by the IKK-NF-κB cascade, whereas interferon-γ (IFN-γ) acts through transcriptional factor STAT1 and IRF-1. Previous studies showed that heat shock protein 90 is essential for iNOS gene transactivation. But the role of the closely related heat shock protein 70 (Hsp70) in iNOS induction remains unknown. We address this issue in cultured cells and endotoxemic mice. With mouse macrophages, Hsp70 inhibition or knockdown prevented LPS/IFN-γ-stimulated iNOS protein expression. RT-PCR experiments showed that both LPS- and IFN-γ-induced iNOS mRNA transcriptions were blocked by Hsp70 inhibitor. The preventing effect of Hsp70 inhibition on iNOS gene transcription was confirmed in vivo in endotoxemic mice. Further studies revealed that Hsp70 inhibition disabled IKK activation in LPS-stimulated cells, hence precluding NF-κB-initiated iNOS gene transcription. Intriguingly, Hsp70 inhibition had little effect on IFN-γ-elicited STAT1 activation or IRF-1 upregulation. But ChIP assays showed that both STAT1 and IRF-1 bindings to iNOS promoters were markedly reduced in Hsp70-inhibited cells. Hsp70 inhibition had no significant effect on iNOS mRNA stability. These studies uncover the necessity of Hsp70 for iNOS induction. Hsp70 is required for IKK activation and STAT1/IRF-1 promoter binding amid iNOS gene transactivation. Selectively targeting Hsp70 may be a new approach to intervene iNOS expression in diseases.