Epithelial-to-mesenchymal transition (EMT) plays an important role in invasion and metastasis of hepatocellular carcinoma (HCC). Our previous study found that atypical protein kinase C-iota (aPKC-iota) promoted the EMT process in HCC. However, how the aPKC-iota signaling pathway is regulated in HCC has not been elucidated. In this study, vector transfection was utilized to study the invasion of HCC cells, and the mechanism between P300 and aPKC-iota signaling pathways in regulating the EMT process of HCC was further elucidated in vitro and in vivo. We found both P300 and aPKC-iota were highly expressed in HCC and they were correlated with tumor progression and poor survival in HCC patients. P300 knockdown inhibited EMT, invasion and other malignant events of HCC cells but promoted cell apoptosis and cycle arrest. However, the effects mediated by P300 knockdown were abolished by aPKC-iota overexpression. Further studies showed that P300 upregulates aPKC-iota expression through increasing the transcription of Elk1, a transcriptional activator of aPKC-iota, and stabilizing Elk1 protein and its phosphorylation. In conclusion, our work uncovered the molecular mechanism by which oncogenic aPKC-iota is upregulated in HCC and suggests that P300, like aPKC-iota, may be used as a prognostic biomarker and therapeutic target in patients with HCC.