Objective To investigate the function of Smad2/3a on vertebrate neural crest cell development.Methods Knock-down of smad2/ 3 and overexpression of casmad2 and smad3a were achieved by microinjection of antisensemorpholino (MO) and mRNA into one-cell stage embryos, respectively. Then, the expression of snail1b, sox10, foxd3, andcrestin in MO-injected embryos and crestin in mRNA-injected embryos at the 6-somite stage were detected using whole-mountin situ hybridization. casmad2 mRNA and smad3a mRNA were used to rescue smad2 and smad3a morphants. Results Knockdown of smad2 and smad3a resulted in a remarkable downregulation of crestin, while there were no obvious changes inthe expression of snail1b, sox10, or foxd3 in the smad2/ 3a morphants. Knockdown of smad3b did not inhibit the expression ofcrestin, snail1b, sox10, or foxd3. Overexpression of casmad2 and smad3a led to an upregulation of crestin. Overexpression ofsmad2 and smad3a restored the reduction of crestin expression caused by smad2 and smad3a depletion. Conclusions Smad2 and Smad3a play key roles in the regulation of expression levels of crestin during neural crest cell development.