The inhibitory roles of limonin have been revealed in various tumors. However, the roles and related mechanism of limonin in hepatocellular carcinoma (HCC) progression are still confused. Here, we collected non-adherent spheroids formed by HCC cells and found that the proportion of spheroids in G0 phase was remarkably higher than that in HCC cells. Additionally, limonin increased EdU incorporation without affecting apoptosis in spheroids. Notably, upon limonin treatment, the proportion of spheroids in G0 was decreased and S/G2/M increased. Furthermore, we found that limonin attenuated the stemness of HCC cells, characterized as the decreased ALDH1 activity, stemness marker expression and spheroid formation ability. Moreover, an integrated transcriptional analysis based on RNA-sequencing data was employed to gain mechanistic insight into limonin functioning, and we found that the most significant pathways identified centered on PI3K/Akt signaling. qRT-PCR and western blot obtained the consistent results. Overall, these data suggest that limonin attenuates the stemness of HCC cells by reducing cellular quiescence through activating PI3K/Akt signaling.