Background Hepatocellular carcinoma (HCC) metastasis and recurrence lead to therapy failure. Posttranslational modification (PTM) mechanisms in HCC, especially those of recently discovered PTMs, such as lysine crotonylation (Kcr), are still poorly understood. Methods Kcr protein levels in HCC cell lines of MHCC-97H (highly invasive) cells and MHCC-97L (minimally invasive) were detected through stable isotope labeling by amino acids in cell culture (SILAC) and liquid chromatography with tandem mass spectrometry (LC–MS/MS). The differentially crotonylated proteins in these two cell lines were identified through bioinformatics analysis. Protein Kcr was confirmed by immunoprecipitation (IP) and western blotting (WB). The function of Septin2 (SEPT2) crotonylated at K74 (SEPT2-K74cr) in metastasis was examined in vivo and in vitro. The clinical significance of SEPT2-K74cr in HCC patients was determined by immunohistochemistry (IHC) assay. Results The crotonylation rate was higher in highly invasive cells, and higher crotonylation level in HCC cells facilitated cell migration and invasion. The crotonylated protein SEPT2 was significantly hypercrotonylated in highly invasive cells, and the de-crotonylated mutation of K74 in SEPT2 impaired SEPT2 GTPase activity and inhibited HCC metastasis in vitro and in vivo. Mechanistically, SIRT2 decrotonylated SEPT2, and P85α was found to be the downstream effector of SEPT2. SEPT2-K74R facilitated P85α degradation, suppressing PI3K pathway and inhibiting the epithelial–mesenchymal transition. SEPT2-K74cr was correlated with poor prognosis and recurrence in HCC patients. Conclusions We revealed the role of nonhistone protein crotonylation in regulating HCC metastasis and invasion. Crotonylation facilitated cell invasion through the crotonylated SEPT2-K74 (cr)-P85α-AKT pathway. High SEPT2-K74 crotonylation predicted poor prognosis and a high recurrence rate in HCC patients.