Inflammation is a hallmark of chlamydial infections, but how inflammatory cytokines are induced is not well understood. Pattern recognition receptors (PRR) of the host innate immune system recognize pathogen molecules and activate intracellular signaling pathways that modulate immune responses. The role of PRR such as Toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD) proteins in the endogenous interleukin-8 (IL-8) response induced during Chlamydia trachomatis infection is not known. We hypothesized that a PRR is essential for the IL-8 response induced by C. trachomatis infection. RNA interference was used to knock down the TLR signaling partner MyD88 as well as NOD1 and its signaling molecule receptor-interacting protein 2 (RIP2). IL-8 induced at 30 h postinfection by C. trachomatis was dependent on NOD1 signaling through RIP2; however, the IL-8 response was independent of MyD88-dependent TLR signaling. Activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cellular signaling pathway, which is essential for up-regulation of IL-8 in response to C. trachomatis infection, was independent of NOD1 or RIP2. We conclude that the endogenous IL-8 response induced by C. trachomatis infection is dependent upon NOD1 PRR signaling through RIP2 as part of a signal system requiring multiple inputs for optimal IL-8 induction. Since ERK is not activated through this pathway, a concomitant interaction between the host and bacteria is additionally required for full activation of the endogenous IL-8 response.