Hepatoma-derived growth factor (HDGF) is a nucleus-targeting mitogen for various types of cells. Besides, HDGF overexpression is associated with tumor progression and poor survival outcome in patients with hepatocellular carcinoma (HCC) and lung cancer. HDGF is capable of promoting the proliferation and migration in various types of cells. HDGF is composed of 240 amino acids and contains 2 putative bipartite nuclear localization signals (NLSs). By dividing HDGF into two deletion domains: PWWP (residues 1-100) and C140 (residues 101-240), we found that both PWWP and C140 domains are capable of promoting nuclear localization, However, only C140 domain promoted cell proliferation and migration as HDGF. Mutation in NLS domains abrogated the nuclear localization and growth-promoting function, but not the migratory potential of HDGF. Beside, Ser165 was predicted as putative cdc2 phosphorylation site. In vitro kinase assay indicated that Ser165 of HDGF is the phosphorylated site of cdc2 kinase. We also demonstrated that mutations in cdc2 phoshprylation site did not affect the nuclear localization, proliferation-stimulating activities of HDGF but enhance migration-stimulating abilities of HDGF. Recently, the HDGF domain containing residues 81 to 100 is shown to be responsible for binding to membrane receptor in NIH3T3 cells. Besides, Lys96 plays a pivotal role for receptor binding. By generation of HDGF Lys96A mutant protein, we found that mutation of Lys96 indeed caused a prominent reduction in cellular binding affinity of HDGF to NIH3T3 cells and affect cell migration. In summary, the NLSs are essential for the mitogenic effect of HDGF, but not required for migration. And the cdc2 phoshorylation site is important for NIH3T3 migration. The Lys96 of HDGF play an important role of membrane receptor binding and cell migration.