Objective To investigate whether rhubarb anthraquinones can activate the insulin signaling pathway in HepG2 cells in the presence and absence of insulin resistance. Methods After an insulin resistance model of HepG2 cells was constructed using aldosterone (ALD), whether rhubarb anthraquinones (including rhein, chrysophanol, emodin and aloe-emodin) at concentrations of 2.5-20 μmol/L could activate the insulin signaling pathway in HepG2 cells was investigated by detecting cellular activity with Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Contents of cellular glucose and glycogen levels were measured with glucose and glycogen content assay kit, respectively, and protein expression levels of phosphorylated protein kinase B/protein kinase B (p-Akt/Akt) and phospho-glycogen synthase kinase3β/glycogen synthase kinase-3β (p-GSK-3β/GSK-3β) were determined with Western blotting. SPSS statistics 20.0 was used for data analysis. Student′s t test or ANOVA was employed for intergroup comparison. Results Rhein, chrysophanol, emodin and aloe-emodin of 2.5-20 μmol/L had no significant effect on the survival of HepG2 cells (P>0.05). However, they increased the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β in the presence and absence of insulin resistance(P<0.05). Further studies also found that the relative glucose content was reduced in the rhein, chrysophanol and emodin treatment groups (P<0.05), but no such change was seen in the aloe-emodin treatment group (P>0.05). The relative glycogen content was decreased in all the rhein, chrysophanol, emodin and aloe-emodin treatment groups (P<0.05). Both rhein and emodin were able to reverse the ALD-induced reduction of glycogen granules in HepG2 cells. Conclusion Rhubarb anthraquinones (rhein, chrysophanol, emodin and aloe-emodin) activate the glycogen synthesis pathway in HepG2 cells in the presence and absence of insulin resistance.