This study explores the anti-inflammatory properties of lupeol, a notable phytosterol found in various medicinal plants, highlighting its potential as a candidate for new drug development. We examined the effects of lupeol on heme oxygenase (HO)-1, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), as well as its impact on inflammatory markers in the lung tissues of LPS-challenged mice. Lupeol treatment enhanced HO-1 production, inhibited nuclear factor (NF)-κB activity, and reduced levels of COX-2/prostaglandin E2 (PGE2) and iNOS/nitric oxide (NO). In addition, lupeol decreased the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) and promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), enhancing its binding to the anti-oxidant response element (ARE) and subsequently reducing interleukin (IL)-1β expression. Invivo, lupeol significantly lowered iNOS expression and tumor necrosis factor (TNF)-α levels in bronchoalveolar lavage fluid from LPS-treated mice. These findings suggest that lupeol exerts its anti-inflammatory effects by modulating key signaling pathways, positioning it as a promising candidate for the development of novel therapeutics targeting pathological inflammation.