Nuclear Factor Kappa-B (NF-κB) is a transcription factor that controls expression of genes involved in the immune and inflammatory responses as well as being a key component in the onset of cancers. In this study, we provided evidence that mitogen- and stress-activated protein kinase (MSK1) is responsible for a noncanonical late-phase activation of NF-κB upon UVB irradiation. Our data demonstrated that following UVB irradiation, MSK1 is activated via phosphorylation at the 24 h time point coinciding with translocation of NF-κB into the nucleus. Investigations into the signaling pathways upstream of MSK1 through the use of specific inhibitors for mitogen-activated protein kinase and p38 revealed that both kinases are required for full phosphorylation during the late phase (24 h), while p38 is paramount for phosphorylation during the early phase (6 h). Electromobility shift assays (EMSA) showed that inhibition of MSK1 resulted in a marked reduction in NF-κB binding affinity without altering the nuclear translocation of NF-κB. Supershift EMSA implicate that the p65, but not p50, isoform of NF-κB is involved in late-phase activation in response to UVB irradiation. Together, the results of these studies shed light onto a novel pathway of MSK1-mediated late-phase activation of NF-κB in response to UVB irradiation.