Members of the transforming growth factor-β (TGF-β) superfamily have significant roles in the regulation of a wide variety of physiological processes. In our present work, phylogenetic tree analysis showed that human GDF3 (Growth and differentiation factor 3) and human GDF1 formed a subgroup of closely related molecules. Through quantitative real-time PCR analysis in different human tissues, GDF1 and GDF3 expression level had a big difference in brain. GDF3 could activate downstream signaling through associating with ALK7 (Activin receptor-like kinase 7) in a Cripto-dependent fashion. A CHO cell line stably transfected with the encoding sequence of GDF3, named CHO-GDF3, was established. Western blotting analysis demonstrated that GDF3 protein could be secreted into the medium from CHO cells and immunofluorescence experiment showed that GDF3 was mainly distributed in cytoplasm of the stable cell line, the primary hippocampal neurons, and brain tissues. Furthermore, the conditioned medium from CHO-GDF3 could reduce PC12 cell growth and induce cell differentiation. All these findings bring new insights into the functional study of GDF3.