Nuclear factor-kappa B (NF-κB) plays an important role in UV-induced skin tumorigenesis. Activation of NF-κB by UV-irradiation is composed of two phases. The early phase culminates with maximal levels of DNA binding ability at 4 h postirradiation and is dependent on translational inhibition. The late-phase activation of NF-κB occurs between 16 and 48 h post-irradiation and the mechanism is not clear due to the fact that NF-κB was activated in the presence of high level of IκBα. In this report, we provide evidence that without translational inhibition, the transcription of IκBα was induced by UV-irradiation. In the late-phase of UV-induced NF-κB activation, the IκBα depletion is the combined result of regulation at both transcriptional and translational levels. Neither ubiquitination nor proteasomal degradation have detectable attributions to IκBα breakdown. We also demonstrate that UV only induced phosphorylation of p65(S276), while tumor necrosis factor-α induced phosphorylation at both Ser276 and 536 sites of p65. Based upon our results, we propose a novel mechanism for translation-regulated IκBα depletion and MSK-mediated NF-κB activation at 24 h post-UV-irradiation.