HCC (hepatocellular carcinoma) is among the most common and lethal cancers worldwide with a poor prognosis mainly due to a high recurrence rate and chemotherapy resistance. ATO (arsenic trioxide) is a multi-target drug that has been effectively used as an anticancer drug in acute promyelocytic leukaemia. However, a Phase II trial involving patients with HCC indicates that the use of arsenic as a single agent is not effective against HCC. TGIF (TG-interacting factor) is a transcriptional co-repressor that interferes with TGF-β (transforming growth factor-β) signalling which plays a growth-inhibitory role in HCC. In the present study, we demonstrated that ATO induced hepatocellular apoptosis via TGF-β/Smad signalling and led to downstream induction of p21(WAF1/CIP1) (p21). However, ATO could also induce TGIF expression via a post-transcriptional regulation mechanism to antagonize this effect. Using a biotin-labelled RNA probe pull-down assay and in vivo RNA immunoprecipitation analysis, we identified that HuR (human antigen R) bound to the TGIF mRNA 3'-UTR (3'-untranslated region) and prevented it from degradation. ATO treatment increased the interaction between HuR and TGIF mRNA, and reduction of HuR expression inhibited ATO-induced TGIF expression. Moreover, the EGFR (epidermal growth factor receptor)/PI3K (phosphoinositide 3-kinase)/Akt pathway was shown to mediate the post-transcriptional regulation of TGIF in response to ATO. Finally, we also demonstrated that the down-regulation of TGIF could sensitize ATO-induced HepG2 cell apoptosis. Collectively, we propose that the EGFR/PI3K/Akt pathway may regulate the post-transcriptional regulation of TGIF expression to antagonize ATO-induced apoptosis in HCC. Blockage of the PI3K/Akt pathway or TGIF expression combined with ATO treatment may be a promising strategy for HCC therapy.