Hca-P and Hca-F is a pair of synogenetic mouse hepatocarcinoma ascites cell lines, possessing different capacity of lymphatic metastasis. Receptor of activated C-kinase 1 (Rack1), together with Jnk1 and gelsolin (Gsn) were previously identified as differentially expressed proteins for lymphatic metastatic potential between the two cell lines. As an intracellular scaffold protein, Rack1 could recruit such signaling molecules as integrins, Src, PKC which are involved in many important biological processes and play key roles in cancer progression. In our present studies, pCDNA3.1(+)-Rack1, a eukaryotic expression plasmid, was constructed and stably transfected into Hca-P cells with a low metastatic potential. CCK8 assay and transwell system were used to evaluate the effects of Rack1 on proliferation, migration and invasion of Hca-P cells in vitro. Then, LY294002, an inhibitor of PI3K, was added into the culture medium of pCDNA3.1(+)-Rack1-Hca-P cells and their biological behaviors observed further. Moreover, the expression of Jnk1, Rac1 and Gsn of pCDNA3.1(+)-Rack1-Hca-P cells were detected by western blot after pretreated with various doses of LY294002. As a result, the proliferation, migration and invasion of pCDNA3.1(+)-Rack1-Hca-P cells were significantly enhanced and could be inhibited by LY294002. In addition, the expression of Gsn, Rac1 and Jnk1 of pCDNA3.1(+)-Rack1-Hca-P cells also decreased after pretreated with LY294002. The expression of Gsn can be inhibited by NSC33766 (an inhibitor of Rac1). Taken together, Rack1/PI3K/Rac1 signaling pathway may play a crucial role in malignant biological behaviors of mouse hepatocarcinoma cells with lymphatic metastasis potential. It may be a potential target for therapy of cancer lymphatic metastasis.