A nerve growth factor-differentiated PC12 cell line was used to investigate the protective effects of s-methyl cysteine (SMC) at 1, 2, 4, and 8 μM under oxygen-glucose deprivation (OGD) conditions. OGD decreased the cell viability. However, SMC pre-treatments at 2, 4 and 8 μM improved the cell viability, decreased cleaved caspase-3 and Bax expression, and reserved Bcl-2 expression. Furthermore, SMC maintained the mitochondrial membrane potential, lowered the intracellular Ca(2+) concentration and DNA fragmentation, and decreased the activity and expression of caspase-3 and caspase-8. OGD increased the reactive oxygen species (ROS) and 3-nitrotyrosine production, decreased glutathione peroxide (GPX) and glutathione reductase (GR) activities and the expression, enhanced nitric oxide synthase (NOS) activity and inducible NOS (iNOS) expression. SMC pre-treatments at 2, 4 and 8 μM lowered the ROS and 3-nitrotyrosine formation, maintained GPX and GR activities and expression, and decreased NOS activity and iNOS expression. OGD up-regulated hypoxia-inducible factor (HIF)-1α, nuclear transcription factor kappa (NF-κ) B p50, NF-κB p65 and p-p38 expression. SMC pre-treatments at 1-8 μM lowered HIF-1α expression and decreased p38 phosphorylation. SMC at 2, 4 and 8 μM suppressed the protein expression of NF-κB p50 and NF-κB p65. When YC-1 (HIF-1α inhibitor), pyrrolidine dithiocarbamate (NF-κB inhibitor) or SB203580 (p38MAPK inhibitor) were used to block the activation of HIF-1α, NF-κB and p38, SMC pre-treatments did not affect the protein expression of HIF-1α, NF-κB and p-p38. These results indicated that SMC was a potent neuro-protective agent.