SummaryNitric oxide (NO) is a potent molecule involved in the cytotoxic effects mediated by macrophages (MØ) against microorganisms. We previously reported that Src homology 2 domain phosphotyrosine phosphatase 1 (SHP‐1)‐deficient cells generate a greater amount of NO than wild‐type cells in response to interferon‐γ (IFN‐γ). We also reported that theLeishmania‐induced MØ SHP‐1 activity is needed for the survival of the parasite within phagocytes through the attenuation of NO‐dependent and NO‐independent mechanisms. In the present study, we investigated the role of SHP‐1 in regulating key signalling molecules important in MØ NO generation. Janus tyrosine kinase 2 (JAK2), mitogen‐activated extracellular signal‐regulated protein kinase kinase (MEK), extracellular signal‐regulated kinases 1 and 2 (Erk1/Erk2) mitogen‐activated protein kinases, p38 and stress‐activated mitogen‐activated protein kinases/c‐Jun NH2‐terminal kinase (SAPK/JNK) were examined in immortalized bone marrow‐derived MØ (BMDM) from both SHP‐1‐deficient motheaten mice (me‐3) and their respective littermates (LM‐1). The results indicated that Erk1/Erk2 and SAPK/JNK are the main kinases regulated by SHP‐1 because the absence of SHP‐1 caused an increase in their phosphorylation. Moreover, only Apigenin, the specific inhibitor of Erk1/Erk2, was able to block IFN‐γ‐induced inducible nitric oxide synthase (iNOS) transcription and translation in me‐3 cells. Transcription factor analyses revealed that in the absence of SHP‐1, activator protein‐1 (AP‐1) was activated. The activation of AP‐1, and not nuclear factor‐κB (NF‐κB) or signal transducer and activator of transcription‐1α (STAT‐1α), may explain the enhanced NO generation in SHP‐1‐deficent cells. These observations emphasize the involvement of the MAPKs Erk1/Erk2 and SAPK/JNK in NO generation via AP‐1 activation. Collectively, our findings suggest that SHP‐1 plays a pivotal role in the negative regulation of signalling events leading to iNOS expression and NO generation. Furthermore, our observations underline the importance of SHP‐1‐mediated negative regulation in maintaining NO homeostasis and thus preventing the abnormal generation of NO that can be detrimental to the host.