Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) is a member of the RNA-binding protein family and emerges as an important gene expression regulator by assisting post-transcriptional modification including alternative splicing of pre-RNA transcripts and shuttling RNA from nuclear to cytoplasm. During mouse embryonic stem (ES) cell differentiation into smooth muscle cells (SMCs) on collagen IV-coated flasks, the upregulation of hnRNP A2/B1 was revealed by proteomic analysis, real-time PCR and Western blot analysis. Enforced expression of hnRNP A2/B1 resulted in increased multiple SMC-specific differentiation genes expressions (Smooth muscle α actin, Smooth Myosin 22α, calponin and Smooth Muscle Myosin Heavy Chain) and transcription factor expression (Serum Response Factor, Myocardin and Myocyte Enhancer Factor 2C) in a dose-dependent manner. On the other hand, siRNA-induced suppression of hnRNP A2/B1 downregulated those genes at both the protein and RNA levels. Moreover, whole mount immunohistochemistry and whole mount in situ hybridisation of hnRNP A2/B1 on chick embryos showed a cardiovascular system related expression pattern during embryogenesis. While immunohistochemical staining on sections displayed that hnRNP A2/B1 localised within vessel walls during the formation of dorsal aorta and was enriched in branchial arches. More importantly, when hnRNP A2/B1 morpholino was induced into chick embryonic neural crest, it subsequently inhibited smooth muscle development in branchial arch arteries. Taken together, these data provide novel information on the role of hnRNP A2/B1 in smooth muscle differentiation, and indicate that it can potentially be a new therapeutic target for the intervention of vascular proliferative diseases.