Abstract In the present study, the in vitro and in vivo anti-acne activation by flavonoid combination therapy were investigated, and the role and relationship between flavonoid and FOXO3 signaling pathway were discussed. Three natural flavonoid such as quercetin, kaempferol, luteolin had certain anti-inflammation activity. Meanwhile, the combination therapy significantly outperforming using these flavonoids alone, which might be attributed to the synergistic bactericidal effect on the cells. Besides, it was also found that the FOXO3 pathway play an important role in P acnes induced skin inflammation. It’s promoted the decreasing of FOXO3 signaling pathway activation and induced an increasing in sebum secretion. During the acne inflammatory response, the expression of pro-inflammatory cytokine was increased significantly. After flavonoid combination therapy, the pro-inflammatory cytokines expression levels such as TNF-α (19.36 ± 2.44 pg/ml), IL-8 (1392.52 ± 131.75 pg/ml), IL-1β (13.42 ± 4.54 pg/ml) and IL-6 (109.64±13.34 pg/ml) were significantly reduced. Flavonoid combination therapy effectively decreased the expression of pro-inflammatory cytokines and increased the secretion of the anti-inflammatory cytokines. In addition, the activation of the FOXO3 pathway was found to be inversely proportional to the expression of pro-inflammatory cytokines. In this study, the flavonoid combination therapy demonstrated a “synergistic effect” inhibiting the inflammatory process via the activation of the FOXO3 pathway. The enhanced synergy observed surpassed their individual effects when used in combination. Consequently, the “synergistic effect” of three flavonoid ingredients suggested a new approach for the development of anti-acne reagents as FOXO3 enhancers.