Objective To investigate whether genistein alleviates alcoholic fatty liver disease (AFLD) by regulating sirtuin1 (SIRT1) / sirtuin3 (SIRT3) / forkhead box protein O1 (FOXO1) signaling pathway. Methods A HepG2 AFLD model was induced using 100 μmol / L oleic acid combined with 150 mmol / L 95% ethanol. The experiment was divided into 16 groups, including control, model, genistein, and resveratrol groups, SIRT1 gene silencing control + model, genistein, and resveratrol groups (C-shSIRT1, M-shSIRT1, G-shSIRT1, and R-shSIRT1,respectively), SIRT3 gene silencing control + model, genistein, and resveratrol groups (C-shSIRT3, M-shSIRT3, GshSIRT3, and R-shSIRT3, respectively), and SIRT1 / SIRT3 dual gene silencing control + model, genistein, and resveratrol groups (C-shSIRT1 / 3, M-shSIRT1 / 3, G-shSIRT1 / 3, and R-shSIRT1 / 3, respectively). Cellular steatosis was analyzed through Oil Red O staining and measurements of triglyceride, total cholesterol, and free fatty acid levels.Intracellular levels of tumor necrosis factor-α and interleukins 6 and 1β were determined by enzyme-linked immunosorbent assay. Western blot was used to detect protein expression levels of SIRT1, SIRT3, and FOXO1, and their interactions were analyzed by co-immunoprecipitation. Results Genistein significantly reduced lipid deposition and inflammatory responses in HepG2 AFLD model cells ( P<0. 05). Compared with the control group, protein expression of SIRT1, SIRT3, and FOXO1 was significantly decreased in the model group (P<0. 01). Compared with the model group, the genistein group showed significantly increased protein expression of SIRT1, SIRT3, and FOXO1 (P<0. 05). Co-immunoprecipitation result showed that SIRT1, SIRT3 and FOXO1 interacted. Genistein significantly reduced cellular steatosis after either SIRT1 or SIRT3 gene silencing, but its anti-AFLD effect was attenuated when both genes were simultaneously silenced. Compared with the C-shSIRT1 group, FOXO1 expression 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. 01). Compared with the M-shSIRT3 group, FOXO1 expression was significantly increased in the G-shSIRT3 group (P<0. 01). Compared with the C-shSIRT1 / 3 group,FOXO1 expression was significantly decreased in the M-shSIRT1 / 3 group (P<0. 01); FOXO1 expression levels were similar between the M-shSIRT1 / 3 and G-shSIRT1 / 3 groups (0. 05). Conclusions Genistein ameliorates lipid metabolism and suppresses inflammatory response in HepG2 AFLD cells by regulating SIRT1 / SIRT3 / FOXO1 signaling pathway, with the SIRT3 / FOXO1 axis playing a particularly crucial role.