It has previously been reported that 16α, 17α-epoxypregnenolone-20-oxime (EPREGO) exerts an inhibitory effect on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in microglia. The present study aimed to investigate the effects of EPREGO on the lipopolysaccharide (LPS)‑induced inflammatory response in RAW264.7 macrophage cells, and to determine the underlying molecular mechanisms using western blot analysis, enzyme‑linked immunosorbent assays and fluorescence‑activated cell sorting. The present study demonstrated that LPS‑induced production of NO and interleukin (IL)-6, and the protein expression levels of iNOS, were reduced by EPREGO in a dose‑ and time‑dependent manner, whereas, EPREGO did not affect tumor necrosis factor‑α production. In addition, EPREGO suppressed LPS‑induced cellular reactive oxygen species production and phagocytosis. Furthermore, EPREGO significantly inhibited the LPS‑induced activation of mitogen‑activated protein kinases and inhibitor of κB α degradation in LPS‑stimulated RAW264.7 cells, thus resulting in modulation of the production of NO and IL‑6. Taken together, these results suggest that EPREGO exhibits anti-inflammatory activity in macrophages, thus validating the hypothesis that EPREGO may be useful as a therapeutic agent for the treatment of macrophage-mediated inflammation.