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 24h of hypoxic exposure results in 20% cell death (IC₂₀) 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.