In vitro gene profiling studies have associated the molecular pathways of Nrf2-Keap1 and Tolllike receptor (TLR) signaling with skin sensitization. In this study, the role of these pathways in the regulation of protein biomarkers for skin sensitization was further elucidated using transient gene knock-down of key components of the signaling cascades in HaCaT cells after exposure to dinitrochlorobenzene (DNCB). The effect of targeting these pathways was established through evaluation of heme oxygenase1 (HMOX1) and interleukin (IL)-8 production. These experiments showed that Nrf2 is not involved in regulating HMOX1 after exposure to DNCB, but that activation of TLR signaling moderates the expression of HMOX1. The regulation of IL-8 depended on Nrf2, but also on the Toll/interleukin-1 receptor (TIR)-domain-containing adapterinducing interferon-(TRIF) adaptor protein in TLR signaling. This study provides new insights into the regulation of HMOX1 and IL-8, but the exact regulating mechanisms remain to be further elucidated.