Objective: To investigate the mechanism of genistein (GEN) in ameliorating alcoholic fatty liver disease (AFLD) through regulation of the Sirtuin1(SIRT1)/Sirtuin3(SIRT3)-Forkhead Box Protein O1(FOXO1) signaling pathway. Methods: An in vitro AFLD model was established by treating HepG2 cells with 100 μmol/L oleic acid combined with 150 mmol/L 95% ethanol. The experiment was divided into 16 groups, including: control group, model group, genistein group, resveratrol group, SIRT1 gene silencing control group (C-shSIRT1), SIRT3 gene silencing control group (C-shSIRT3), SIRT1 gene silencing model group (M-shSIRT1), SIRT3 gene silencing model group (M-shSIRT3), SIRT1 gene silencing genistein group (G-shSIRT1), SIRT3 gene silencing genistein group (G-shSIRT3), SIRT1 gene silencing resveratrol group (R-shSIRT1), SIRT3 gene silencing resveratrol group (R-shSIRT3), SIRT1/SIRT3 double gene silencing control group (C-shSIRT1/3), SIRT1/SIRT3 double gene silencing model group (M-shSIRT1/3), SIRT1/SIRT3 double gene silencing genistein group (G-shSIRT1/3), and SIRT1/SIRT3 double gene silencing resveratrol group (R-shSIRT1/3). The optimal intervention concentrations of genistein and resveratrol were screened by CCK-8 assay. Cellular steatosis was analyzed through Oil Red O staining and measurements of triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) levels. Intracellular levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were determined by ELISA. Western blot was used to detect the protein expression levels of SIRT1, SIRT3, and FOXO1 in each experimental group, and the interactions among these three proteins were analyzed by co-immunoprecipitation (Co-IP) technique. Results: Genistein significantly ameliorated lipid deposition and inflammatory response in HepG2 AFLD model cells (P<0.05). Compared with the control group, the protein expressions of SIRT1, SIRT3 and FOXO1 were significantly decreased in the model group (P<0.01); while compared with the model group, the genistein group showed significantly increased protein expressions of SIRT1, SIRT3 and FOXO1 (P<0.05). The Co-IP results showed that SIRT1, SIRT3 and FoxO1 interacted with each other. Genistein significantly ameliorated cellular steatosis after either SIRT1 or SIRT3 gene silencing, but its anti-AFLD effect was markedly attenuated when both SIRT1 and SIRT3 genes were simultaneously silenced. Compared with the C-shSIRT1 group, FOXO1 expression level was significantly increased in the M-shSIRT1 group (P<0.01). Compared with the M-shSIRT1 group, FOXO1 expression was significantly decreased in the G-shSIRT1 group (P<0.01). Compared with the C-shSIRT3 group, FOXO1 expression was significantly decreased in the M-shSIRT3 group (P<0.0001). Compared with the M-shSIRT3 group, FOXO1 expression was significantly increased in the G-shSIRT3 group (P<0.0001). Compared with the C-shSIRT1/3 group, FOXO1 expression was significantly decreased in the M-shSIRT1/3 group (P<0.001); FOXO1 expression levels were similar between the M-shSIRT1/3 group and G-shSIRT1/3 group (P>0.05). Conclusions: Genistein ameliorates lipid metabolism and suppresses inflammatory response in HepG2 AFLD cells by regulating the SIRT1/SIRT3-FOXO1 signaling pathway, especially suggesting that regulating SIRT3-FOXO1 signaling plays a significant role in genistein anti-AFLD.