The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon-gamma (IFN-γ)-treated HaCaT keratinocytes. We found that IFN-γ-induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N-acetyl cysteine (NAC), significantly inhibited the IFN-γ-induced production of chemokines as well as the activation of I kappa-B (IκB)-nuclear factor-kappa B (NF-κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen-activated kinase (MAPK), and NF-κB suppressed IFN-γ-induced production of TARC and MDC. NF-κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN-γ-stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L-buthionine sulfoximine (L-BSO). However, IFN-γ-stimulated activation of IκB and NF-κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN-γ-stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN-γ-stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF-κB activation.