Pim-3 is a member of proto-oncogene Pim family that encodes serine/threonine kinases. Pim proteins regulate both apoptosis and cellular metabolism by phosphorylating their substrates. Here, we report for the first time that Pim-3 is highly expressed at mRNA and protein levels in endothelial cells (ECs). We found that Pim-3 is concentrated at the cellular lamellipodia and co-localized with focal adhesion kinase (FAK). Pim-3 was dispersed from lamellipodia when ECs were treated with cytochalasin D, an inhibitor of actin polymerization. In addition, small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired EC spreading, migration, and proliferation, leading to a reduction in tube-like structure formation in a Matrigel assay. These results provide the novel evidence that Pim-3 plays an essential role in EC spreading and migration, suggesting that Pim-3 may be an important molecular target for the development of small-molecule inhibitors of angiogenesis. J. Cell. Physiol. 220: 82–90, 2009. © 2009 Wiley-Liss, Inc.