The immunosuppressive activity of TGF-β-mediated signaling is well documented, but in contrast, its ability to promote proinflammatory responses is less clear. In this study, we report that blockade of TGF-β signaling by a specific inhibitor of the TGF-β receptor I [activin receptor-like kinase 5 (ALK5)] SB431542 significantly reduces the production of TNF-α, a key proinflammatory cytokine, by LPS-stimulated human monocyte-derived macrophages. ALK5 protein was only detectable after LPS stimulation, and the failure of treatment with SB431542 to alter TNF-α mRNA expression indicates that regulation is post-transcriptional. The additive effect of blocking TGF-β and p38 MAPK signaling on reducing TNF-α but not IL-6 production suggests that there is selectivity in pathway signaling. SB431542 had similar inhibitory effects on TNF-α production by human monocytes and endothelial cells as well as macrophages. Furthermore, treatment with SB431542 reduced plasma TNF-α levels and tissue damage and thereby, prevented the lethal effects of LPS in a mouse model of septic shock. Our data demonstrate a direct effect of TGF-β signaling via ALK5 on the regulation of TNF-α synthesis.