Binding of bacterial LPS to the Toll-like receptor 4 (TLR4) complex of inner medullary collecting duct (IMCD) cells plays a central role in recognition of ascending bacterial infections and activation of proinflammatory responses. Since proinflammatory cyclooxygenase (COX)-2 is induced in IMCD cells upon LPS exposure, the present study addressed the question of whether TLR4 mediates COX-2 induction in IMCD cells and characterized the underlying signaling mechanisms. Enhanced COX-2 expression and activity in the presence of LPS was diminished by TLR4 inhibition. LPS induced a TLR4-dependent stimulation of NF-κB and the MAPKs p38, ERK1/2, and JNK. Activation of NF-κB was under negative control of JNK, as inhibition of JNK increased NF-κB activity and COX-2 expression. Phosphorylation of p38 and ERK1/2 required TLR4-dependent release of TGF-α with subsequent activation of the epidermal growth factor receptor (EGFR), whereas JNK activation was EGFR independent. Inhibition of p38 or ERK1/2 had no significant effect on LPS-induced NF-κB activation, nor on activator protein 1-, cAMP response element-, or serum response element-driven reporter constructs. However, the transcriptional regulator SP-1 appears to contribute to COX-2 expression after LPS exposure. In conclusion, these results propose that LPS mediates enhanced COX-2 expression in IMCD cells by 1) TLR4-mediated activation of the NF-κB signaling pathway, 2) TLR4-dependent release of TGF-α with subsequent activation of the EGFR and downstream MAPKs p38 and ERK1/2, and 3) TLR4-mediated, EGFR-independent activation of JNK that negatively regulates NF-κB activation.