In previous reports from our group, the protective action of RGZ against hepatic steatosis is by coordinated regulation of adiponectin, Sirt6, and AMPK. Up-regulation of Sirt1/6 and the downstream target genes of Sirt1/6 as well as activation of LKB1/AMPK by RGZ led us to hypothesize that RGZ may exert its positive Diacerein effects by acting on both Sirt1 and Sirt6. From these observations and accumulating evidence of sirtuins and their metabolic regulation, we speculate that Sirt1 and Sirt6 possess similarities in cellular localization and metabolic functions and question whether both sirtuins are synergistic or compensatory. To determine whether Sirt1 and Sirt6 are causally related to the development of hepatic steatosis and its amelioration by RGZ, and whether their actions are synergistic, we directly inhibited the Sirt1/6 pathway using RNAi-mediated gene silencing targeting Sirt1 and/or Sirt6 in AML12 mouse hepatocytes. Sirt1/6 knockdown aggravated hepatocyte fat accumulation as shown by increased TG content and suppressed the favorable effects of RGZ on hepatocyte steatosis. In addition, Sirt1/6 knockdown suppressed gene expression of Ppargc1a and Foxo1 as expected, and abolished the RGZ-mediated activation of LBK1 and AMPK. However, double knockdown of Sirt1 and Sirt6 did not result in a synergistic effect on the RGZ-mediated alterations. Conventionally, it has been considered that Sirt1 and Sirt6 exert their functions as NAD -dependent deacetylase and ADP-ribosyltransferase. However, provocative reports have suggested that Sirt1 may be responsible for transcriptional regulation of downstream target genes. Sirt1 upregulated adiponectin gene expression in fully differentiated 3T3-L1 adipocytes by enhancing Foxo1 and C/enhancer-binding protein alpha interaction with the adiponectin promoter. A positive correlation between Sirt1 and Sirt6 has been reported in mice, and, additionally, the increase in Sirt1 occurred earlier than that of Sirt6 in the mouse liver during fasting. Additionally, whether Sirt1 is responsible for transcriptional regulation of Sirt6 was investigated, and the results showed that Sirt1 induced Sirt6 gene expression by forming a complex with Foxo3a and nuclear respiratory factor 1-binding sites on the Sirt6 promoter. Based on these observations, we speculated that Sirt1 and Sirt6 may have synergistic effects on metabolic regulation. However, no synergistic effect between Sirt1 and Sirt6 was observed in this study, suggesting that the relationship between Sirt1 and Sirt6 may be compensatory as a back-up system. Although we could not demonstrate the synergistic effects of two isoforms of sirtuins, our results suggest the possible interaction between Sirt1 and Sirt6. In normal AMP12 hepatocytes without PA 3,4,5-Trimethoxyphenylacetic acid treatment, Sirt1 mRNA expression was not altered by Sirt6 knockdown, but, Sirt6 mRNA expression tends to be lowered by Sirt1 knockdown. These results support the previous findings showing that Sirt1 is responsible for transcriptional regulation of Sirt6. In addition, regarding the effects on Ppargc1a and Foxo1, Sirt1 knockdown resulted in more profound suppression than Sirt6 knockdown. In a hepatocyte steatosis model with PA treatment, the regulatory effects of RGZ on Sirt1 were significantly blunted by Sirt6 knockdown, which suggests that upregulation of Sirt1 by RGZ was partially dependent on Sirt6. On the other hand, RGZ’s effects on Sirt6 were not altered by Sirt1 knockdown in a hepatocyte steatosis model.