Autophagy plays a protective role in endotoxemic mice. Heat shock factor 1 (HSF-1) also plays a crucial protective role in endotoxemic mice by decreasing inflammatory cytokines. The purpose of this study was to determine whether HSF-1 is involved in attenuating the release of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated mice and peritoneal macrophages (PMs) through regulating autophagy activity. Autophagosome formation in HSF-1+/+ and HSF-1-/- mice and PMs stimulated by LPS was examined by Western blotting and immunofluorescence. Lipopolysaccharide-induced autophagy and inflammatory cytokines were examined in HSF-1+/+ and HSF-1-/- PMs treated with 3-methyladenine (3-MA) or rapamycin. Results showed that LPS-induced autophagy was elevated transiently at 12 h but declined at 24 h in the livers and lungs of mice. Higher levels of inflammatory cytokines and lower autophagy activity were detected in HSF-1-/- mice and PMs compared with HSF-1+/+ mice and PMs. Interestingly, LPS-induced release of inflammatory cytokines did not further increase in HSF-1-/- PMs treated with 3-MA but aggravated in HSF-1+/+ PMs. Lipopolysaccharide-induced autophagy did not decrease in HSF-1-/- PMs treated with 3-MA but decreased in HSF-1+/+ PMs. Taken together, our results suggested that HSF-1 attenuated the release of inflammatory cytokines induced by LPS by regulating autophagy activity.