The activation of p38α, a MAPK family member, is associated with macrophage activation by microbial pattern molecules, such as LPS. The requirement of p38α in inflammatory responses has been shown in a number of studies using chemical inhibitors, though the inhibitors also inhibit p38β and perhaps some other enzymes. In this study, we used conditional knockout of p38α in macrophages to address the role of p38α in macrophage activation. We found that p38α deficiency causes a significant inhibition in the production of LPS-induced TNF-α, IL-12, and IL-18, but it has little or no effect on IL-6 or IFN-β production. Knockout of p38α in macrophages did not affect LPS-induced activation of the other major signaling pathways (NF-κB, Jnk, and Erk), nor did it affect the transcriptional activity of NF-κB. It had little inhibitory effect on LPS-induced AP-1 activity, but it significantly inhibited LPS-induced C/EBP-β and CREB activation, indicating that the role of p38α in cytokine production in macrophages is at least in part through its regulation of C/EBP-β and CREB activation. In addition, we also confirmed that p38α is important for phagocytosis of bacteria by macrophages. Our in vivo studies with two murine models showed that p38α is involved in sepsis. Collectively, our data demonstrate that p38α is an important player in inflammatory responses.