LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1alpha (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.