Mammalian ultraviolet (UV) radiation response is a gene induction cascade activated by several transcription factors, including NF-κB. Although NF-κB is induced by UV radiation, the signal transduction mechanism remains relatively unclear. In the present study, we show that UV-induced NF-κB activation is mediated by the activation of Ataxia telangiecia mutated (ATM) and protein kinase C (PKC). We also show that caffeine specifically inhibits UV-mediated NF-κB activation, but not TNFα-mediated NF-κB activation. In addition, our study shows that ATM, but not ATM-Rad3-related (ATR) or DNA-dependent protein kinase (DNA-PK) is involved in UV-induced NF-κB activation. Because SB203580 (a p38 MAPK inhibitor), or Calphostin C or rottlerin (PKC inhibitors) was able to inhibit UV-mediated NF-κB activation, we evaluated whether caffeine could inhibit p38 MAPK or PKC activity. Caffeine or rottlerin inhibited UV-induced phosphorylation of p38 MAPK, but not anisomycin-induced phosphorylation of p38 MAPK, suggesting that p38 MAPK is downstream of PKC. Additionally, caffeine could effectively inhibit UV-induced increases in PKC activity. Taken together, our study demonstrates that caffeine is a potent inhibitor of UV-induced NF-κB activation. Additionally, this inhibition occurs due to the inhibitory action of caffeine on ATM and PKC, resulting in the inhibition of p38 MAPK activation.