Neferine, an alkaloid from N. nucifera has a broad range of pharmacological activity. Hypoxia mediated stress is involved in the generation of inflammatory responses and cell death. The present study evaluated the protective effect of neferine against hypoxia-induced cytotoxicity, oxidative stress and inflammatory response in human Peripheral Blood Mononuclear Cells (hPBMC). Cytotoxicity, as determined by MTT, LDH and NO assays revealed that 24 h of hypoxic exposure results in 20% cell death (IC ₂₀ ) and compromising of cellular integrity, which was restored to near control values by pretreatment with neferine. Oxidative stress parameters such as lipid peroxidation and antioxidant enzymes indicated that neferine exerted a protective effect on hypoxia-induced oxidative stress. Hypoxia-induced Tumor Necrosis Factor-α (TNF-α), Interleukin 6 (IL-6), and Interleukin 8 (IL-8) release were significantly reduced in the neferine pretreated samples indicating its anti-inflammatory role. Our results demonstrate for the first time that neferine exerts a cytoprotective effect against hypoxia-induced oxidative stress and inflammation in hPBMC.