We recently demonstrated that 17β‐estradiol (E2) enhances killing of Leishmania mexicana in macrophages from both male and female DBA/2 mouse by increasing nitric oxide (NO) production. Here, we analyzed the effect of E2 on leishmanicidal activity and cytokine production by bone marrow‐derived macrophages (BMDMs) from male and female C57BL/6 mice in vitro, specifically examining the role of phosphatidylinositol‐3‐kinase‐γ (PI3Kγ) in E2‐induced parasite killing. Unlike its effect on macrophages from both male and female DBA/2 mice, E2 only increased leishmanicidal activity in macrophages from female C57BL/6 mice, which was evident by a significant reduction in both infection rates and infection levels compared to sham controls. E2‐treated BMDMs from female C57BL/6 mice expressed higher levels of interferon‐γRα, and also produced more interleukin (IL)‐12, IL‐6 and NO than both the sham controls and E2‐treated male‐derived macrophages. Sham‐treated BMDMs from female PI3Kγ−/− C57BL/6 mice displayed lower infection rates and infection levels compared to sham‐treated wild‐type (WT) macrophages. However E2, unlike its effect on macrophages from female WT C57BL/6 mice, failed to reduce infection rates and infection levels in BMDMs from female PI3Kγ−/− mice. Interestingly, E2‐treated BMDMs from female C57BL/6 mice produced significant amounts of inflammatory cytokines and NO in levels comparable to those observed in sham‐treated PI3Kγ‐deficient macrophages as well as E2‐treated macrophages from WT mice. These findings show that E2 exerts a distinct effect on leishmanicidal activity of macrophages from male versus female C57BL/6 mice. In addition, they suggest that PI3Kγ is not required for E2‐induced cytokine and NO production in L. mexicana‐infected macrophages from female C57BL/6 mice but it may be involved in parasite clearance from these cells.