Glycogen synthase kinase-3 beta (GSK3β) was initially identified as a key protein in glucose metabolism. GSK3β might be involved in cell growth, motility and apoptosis. Systematic identification of GSK3β-associated proteins is crucial for the exhaustive understanding of its functions. Using GST pull-down experiment and LCMS/MS analysis coupled to bioinformatics tools, we have identified 114 proteins that interacted with GSK3β1 in hepatocellular carcinoma HepG2 cells. Most of the identified proteins are implicated in metabolic process, whereas other proteins are important for cell proliferation or migration, and have been associated with cancer development and metastasis. Several representative proteins, such as hnRNPK, PCNA, Ezrin and STAT1, have been confirmed to interact with GSK-3β1 by co-immunoprecipitation in HepG2 cells. Further studies of these interactions may discover the precise roles and the underlying mechanisms of GSK-3β1 in tumour growth and metastasis.