The homeobox transcription factor Prox1 plays an important role in the development of many embryonic organs. Previous studies indicated that Prox1 facilitates hepatic progenitor-cells proliferation. However, the underlying mechanism of Prox1 in tumor genesis, formation, and progression are poorly understood and need to be exploited. Herein, Chinese Hamster Ovary (CHO) cells were transfected and over-expressed human recombinant Prox1 gene, and developed several stable cell lines of Prox1-CHO after screening. The results indicated that over expression of Prox1 increased CHO cell proliferation in comparison to GFP-CHO and parental CHO cells, and Prox1 increased Akt phosphorylation and up-regulated PI3 Kinase expression. An Akt specific inhibitor-AKTi-X (5 μM) and a PI3 K inhibitor-LY294002 (5 μM) were able to reverse AKT phosphorylation and PI3 K expression induced by Prox1, respectively. Furthermore, AKTi-X but LY-294002 decreased Prox1-CHO cell proliferations at 48 and 72 h. Our results suggest that over expression of Prox1 facilitates CHO cell proliferation via activation of the Akt signaling pathway. This finding provides new insights into the mechanism of Prox1 mediated tumor growth and metastasis where Prox1 is rich.