We used high-throughput Western blotting to identify proteins that are up- or down-regulated by neuronal hypoxia in vitro. Exposure to hypoxia for 24 h produced ≥1.5-fold increases in the expression of 10/700 proteins (1.4%) and decreases in the expression of 16/700 proteins (2.3%). Up-regulated proteins included Arc, doublecortin/calmodulin kinase-like 1, integrin αv, and fibronectin; down-regulated proteins included nuclear autoantigenic sperm protein, protein kinase C–related kinase 2, and E2F transcription factor 1. The prominence of cytoskeleton-related proteins among those showing altered expression highlights the role of the cytoskeleton in neuronal responses to hypoxia.