Resistance to anoikis, cell death due to matrix detachment, is acquired during tumor progression. The 14-3-3 sigma protein is implicated in the development of chemo- and radiation resistance, indicating a poor prognosis in multiple human cancers. However, its function in anoikis resistance and metastasis in hepatocellular carcinoma (HCC) is currently unknown. Methods: Protein expression levels of 14-3-3 sigma were measured in paired HCC and normal tissue samples using western blot and immunohistochemical (IHC) staining. Statistical analysis was performed to evaluate the clinical correlation between 14-3-3 sigma expression, clinicopathological features, and overall survival. Artificial modulation of 14-3-3 sigma (downregulation and overexpression) was performed to explore the role of 14-3-3 sigma in HCC anoikis resistance and tumor metastasis in vitro and in vivo. Association of 14-3-3 sigma with epidermal growth factor receptor (EGFR) was assayed by co-immunoprecipitation. Effects of ectopic 14-3-3 sigma expression or knockdown on EGFR signaling, ligand-induced EGFR degradation and ubiquitination were examined using immunoblotting and co-immunoprecipitation, immunofluorescence staining, and flow cytometry analysis. The levels of EGFR ubiquitination, the interaction between EGFR and 14-3-3 sigma, and the association of EGFR with c-Cbl after EGF stimulation, in 14-3-3 sigma overexpressing or knockdown cells were examined to elucidate the mechanism by which 14-3-3 sigma inhibits EGFR degradation. Using gain-of-function or loss-of-function strategies, we further investigated the role of the EGFR signaling pathway and its downstream target machinery in 14-3-3 sigma-mediated anoikis resistance of HCC cells. Results: We demonstrated that 14-3-3 sigma was upregulated in HCC tissues, whereby its overexpression was correlated with aggressive clinicopathological features and a poor prognosis. In vitro and in vivo experiments indicated that 14-3-3 sigma promoted anoikis resistance and metastasis of HCC cells. Mechanistically, we show that 14-3-3 sigma can interact with EGFR and significantly inhibit EGF-induced degradation of EGFR, stabilizing the activated receptor, and therefore prolong the activation of EGFR signaling. We demonstrated that 14-3-3 sigma downregulated ligand-induced EGFR degradation by inhibiting EGFR-c-Cbl association and subsequent c-Cbl-mediated EGFR ubiquitination. We further verified that activation of the ERK1/2 pathway was responsible for 14-3-3 sigma-mediated anoikis resistance of HCC cells. Moreover, EGFR inactivation could reverse the 14-3-3 sigma-mediated effects on ERK1/2 phosphorylation and anoikis resistance. Expression of 14-3-3 sigma and EGFR were found to be positively correlated in human HCC tissues. Conclusions: Our results indicate that 14-3-3 sigma plays a pivotal role in the anoikis resistance and metastasis of HCC cells, presumably by inhibiting EGFR degradation and regulating the activation of the EGFR-dependent ERK1/2 pathway. To our best knowledge, this is the first report of the role of 14-3-3 sigma in the anoikis resistance of HCC cells, offering new research directions for the treatment of metastatic cancer by targeting 14-3-3 sigma.