Activation of the IκB kinase (IKK) complex by LPS induces phosphorylation and degradation of IκBα, leading to the nuclear translocation of NF-κB. Although it is essential for NF-κB activation, emerging evidence has indicated that the nuclear translocation of NF-κB is not sufficient to activate NF-κB-dependent transcription. Here, we reported that LPS induced the phosphorylation of the p65 trans-activation domain on serine 536 in monocytes/macrophages. Using mouse embryonic fibroblasts lacking either IKKα or IKKβ, we found that IKKβ played an essential role in LPS-induced p65 phosphorylation on serine 536, while IKKα was partially required for the p65 phosphorylation. The LPS-induced p65 phosphorylation on serine 536 was independent of the phosphatidylinositol 3′-kinase/Akt signaling pathway. Furthermore, we found that the phosphorylation on serine 536 increased the p65 transcription activity. In summary, our results demonstrate that IKKβ plays an essential role in the LPS-induced p65 phosphorylation on serine 536, which may represent a mechanism to regulate the NF-κB transcription activity by LPS.