To reveal molecular mechanisms responsible for that over-expression of heat shock protein 27 (HSP27) is associated with poor prognosis in liver cancer, expression of HSP27 was analyzed by RT-PCR and Western blot in Hep3B, MHCC97L and MHCC97H, which had higher metastasis potential than Hep3B, and after HSP27 RNA interference, MHCC97H migratory and invasive capability were evaluated by in vitro migration and invasion assay, apoptosis ratio was detected by flow cytometry(FCM) and TUNEL method, prominent altered signal pathway was analyzed through a Human Q Series Signal Transduction in Cancer Gene Array. The results showed that the up-regulation of HSP27 expression in HCC cell lines was consistent with metastasis potentials. Using RNA interference technique, mRNA and protein level of HSP27 in MHCC97H cell line were specifically reduced by up to 80%. In vitro migration and invasion assay showed the average migratory and invaded cell numbers per field were 21.36 ± 2.92, 19.88 ± 2.23, 11.40 ± 2.05 and 26.35 ± 3.29, 24.43 ± 3.17, 10.92 ± 2.27 respectively in MHCC97H, control RNAi, RNAi groups. Moreover, MHCC97H cell apoptosis ration was 15.12%, 17.56%, 27.64% (MHCC97H, control RNAi, RNAi) respectively by FCM analysis, which was reconfirmed by TUNEL morphological assessment. Furthermore cDNA microarray analysis revealed that NF-κB pathway activation was inhibited after HSP27 RNAi. Immunoblotting analysis showed that the decreasing of nuclear activated NF-κB p65 and phosphorylated IκBα, meanwhile reducing of the association between IKKβ and IKKα in MHCC97H cells after HSP27 RNAi, further co-immunoprecipitation assay showed interaction of IKKβ, IκBα with HSP27 in three HCC cell lines. Altogether, these findings revealed possible roles of HSP27 in being of HCC cell lines metastatic potentials through involving in cellular NF-κB pathway activation.