Macrophages and dendritic cells are required for initiating innate immunity and adaptive immunity. Aberrant activation of macrophages and dendritic cells can cause detrimental immune responses; thus, agents effectively modulating their functions are of great clinical value. We herein investigated whether fisetin, a flavonoid prevalently present in fruits and vegetables, could inhibit macrophage activation and dendritic cell maturation. Fisetin suppressed LPS-induced NF-κB activation, expression of pro-inflammatory proteins (TNF-α and iNOS), MMP-9 activity, and phagocytic activity in macrophages. Furthermore, upon LPS-induced dendritic cell maturation, fisetin at nontoxic concentrations suppressed the expression of costimulatory molecules (CD80 and CD86), the production of cytokines (IL-12, IL-6, and TNF-α), and the endocytic activity of dendritic cells. Fisetin treatment significantly attenuated migration of dendritic cells into spleens and dendritic cell-mediated T cell activation in LPS-treated mice. Collectively, our data reveal that fisetin inhibits macrophage activation and impairs functional maturation of dendritic cells.