Tumor necrosis factor-α (TNFα) and thrombospondin-1 (TSP-1) are well-known mediators of inflammation. However, a causal relationship between TNFα stimuli and TSP-1 expression in endothelial cell stress, and the underlying mechanisms has not yet been investigated. In our study, human microvascular endothelial cells (hMEC) were treated with TNFα and analyzed for endothelial dysfunction, TSP-1 expression, and associated mechanisms. TNFα treatment induced a dose-dependent increase in TSP-1 expression in hMEC associated with increased endothelial permeability, apoptosis, and reduced proliferation. Whereas TNFα activated Akt, ERK, and P38 mitogen-activated protein kinase (P38 MAPK) simultaneously in hMEC, inhibitors of Akt and P38 MAPK, but not ERK blunted TNFα-induced TSP-1 expression. Silencing of NFκB gene had no significant effect on TNFα-induced TSP-1 expression. Our study demonstrates the novel role of TNFα in inducing inflammatory stress response in hMEC through Akt- and P38 MAPK-mediated expression of TSP-1, independent of NFκB signaling.