The role of the p38 signaling pathway in the innate and adaptive immune responses has been well documented, especially in inflammatory cytokine production by dendritic cells (DCs). However, whether the p38 signaling pathway affects the important antigen (Ag) presentation function of DCs remains largely unknown. In this study, we reported that the deletion of p38α resulted in an impaired cross-presentation ability of CD8+ conventional DCs (cDCs) and a reduction in the direct presentation ability of CD8- cDCs ex vivo. Further study revealed that p38α had a crucial role in Ag processing by CD8+ cDCs but did not affect the Ag uptake or co-stimulation of T cells. Moreover, p38α deficiency led to reduced cross-priming of T cells in vivo. The production of the IL-12p40 and IL-12p70 cytokines by p38α-deficient cDCs was also significantly reduced. Our study identified a new role for p38α in modulating the important antigen cross-presentation function of DCs.