Abstract Background Fatty acid synthase (FASN) is highly expressed in various types of cancer and has an important role in carcinogenesis and metastasis. To clarify the mechanisms of FASN in liver cancer invasion and metastasis, the FASN protein interaction network in liver cancer was identified by targeted proteomic analysis. Methods Wound healing and Transwell assays was performed to observe the effect of FASN during migration and invasion in liver cancer. Isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry were used to identify proteins interacting with FASN in HepG2 cells. Differential expressed proteins were validated by co-immunoprecipitation, western blot analyses and confocal microscopy. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to demonstrate the mechanism of FASN regulating metastasis. Results FASN knockdown inhibited migration and invasion of HepG2 and SMMC7721 cells. A total of, 79 proteins interacting with FASN were identified. Additionally, gene ontology term enrichment analysis indicated that the majority of biological regulation and cellular processes that the FASN-interacting proteins were associated with. Co-precipitation and co-localization of FASN with fascin actin-bundling protein 1 (FSCN1), signal-induced proliferation-associated 1 (SIPA1), spectrin β, non-erythrocytic 1 (SPTBN1) and CD59 were evaluated. Knockdown of FASN in liver cancer reduced the expression of FSCN1, SIPA1, SPTBN1 and CD59. Furthermore, inhibition of FASN, FSCN1 or SPTBN1 expression in liver cancer resulted in alterations of epithelial–mesenchymal transition (EMT)-associated markers E-cadherin, N-cadherin, vimentin and transcription factors, Snail and Twist, at the mRNA level, and changes in matrix metallopeptidase (MMP)-2 and MMP-9 protein expression. Conclusion The results suggested that the FASN-interacting protein network produced by iTRAQ-based proteomic analyses may be involved in regulating invasion and metastasis in liver cancer by influencing EMT and the function of MMPs.