In vertebrates, Prox1 is homologous to the Drosophila transcription factor, prospero gene. In order to explore Prox1 expression pattern in goldfish, the 2832bp full-length cDNA was cloned, which encodes a protein with 739 amino acids, and the protein exhibits a 93% similarity to Danio rerio Prox1. The results of RT-PCR and Western-blotting analysis revealed Prox1 expression in eye, brain, heart, liver, spleen and kidney, but not in the muscle of the goldfish. RT-PCR results indicated that there exists a small quantity of maternal Prox1 mRNAs in the mature eggs, and the zygotic Prox1 begins to transcribe from the gastrula embryos, and the expression level increased in the developmental process. Whole-mount in situ hybridization demonstrated that in the eye Prox1 mRNA is first predominately detected in the lens placode at lens stage. Then the Prox1 mRNA is localized in the whole puerile lens cells and retina germinative zone at the heartbeat stage. After lens fibers form, it can be detected predominantly in the optic fiber layer and inner plexiform layer. At the same time, the Prox1 protein expression in the lens is detected by immunofluorescence. At the heartbeat stage, the localization of Prox1 protein is the same as the mRNA. But when the lens fibers are forming, it is detected in a ring-shaped region on lens fibers under lens epithelium, which is different from the mRNA localization. The results suggest that the Prox1 gene plays an important role during lens development, and the roles are different at different developmental stages. In contrast to expression patterns from the outside to the inside of the mouse, the Prox1 localization in goldfish lens exhibits from inside to outside.