Although it is known that smoking during pregnancy induces fetal malformations, few basic studies at the molecular level are currently available. Since it is known that neural crest cells (NCC) play an important role in tissue development and differentiation, we investigated the influence of cigarette smoke extract (CSE) on NCC migration. CSE treatment reduced the migration index of NCC in dose- and tar-content-dependent manners without induction of apoptosis or decrease in proliferation of NCC. alpha-Naphthoflavone, an antagonist of aryl hydrocarbon receptor (AhR), prevented the reduction in NCC migration that was otherwise induced by CSE treatment. Overexpression of AhR caused a significant decrease in NCC migration index, implying that CSE can attenuate NCC migration through AhR signaling. Transcriptome analysis revealed that overexpression of AhR led to decreased expression of R-spondin1 in NCC. Furthermore, overexpression of R-spondin1 prevented the inhibitory effect of CSE on NCC. These results suggest that CSE causes suppressed expression of R-spondin1 by activating signals via the AhR, which leads to impaired neural crest cell migration.