<strong><em>Aims:</em></strong> Hsp27, a master molecular chaperone, plays an important role in cancer. However, the specific co-chaperones that partner with Hsp27 and the role of Hsp27 in hepatocellular carcinoma (HCC) are not fully enumerated. The present study focuses on the role of Hsp27 in HCC and explores its potential co-chaperones in HCC development. <b><i>Methods:</i></b> Gene overexpression or knockdown was used to observe the role of Hsp27 in HCC. Co-immunoprecipitation and mass spectrometry were used to explore apoptosis resistance by regulating multiple co-chaperones of Hsp27. Hsp27 protein-protein interaction (PPI) networks were constructed by the MetaCore software. <b><i>Results:</i></b> Hsp27 was upregulated in HCC tissues, and Hsp27 overexpression significantly facilitated formation of HCC cell colony and invasion in normoxia and tolerance in hypoxia by interacting with HIF-1α. Next, the analysis of microarrays revealed that Hsp27 regulated several cellular signaling pathways, including Wnt, ErbB and TGF-β signaling. Moreover, we characterized the Hsp27 PPI map, which indicated that Hsp27 along with its co-chaperones formed different complexes and exerts transcription regulation activity by activating sp1, c-Myc, p53 and ESR1. <b><i>Conclusions:</i></b> Hsp27 along with its co-chaperones was related to the development of HCC by regulating multiple signaling pathways, and drugs that target Hsp27 along with its co-chaperones may be a potential therapy for HCC.<br
