The Hox code in the neural crest cells plays an important role in the development of the complex craniofacial structures that are characteristic of vertebrates. Previously, 3'AmphiHox1 flanking region has been shown to drive gene expression in neural tubes and neural crest cells in a retinoic acid (RA)-dependent manner. In the present study, we found that the DR5-type RA response elements located at the 3' AmphiHox1 flanking region of Branchiostoma floridae are necessary and sufficient to express reporter genes in both the neural tube and neural crest cells of chick embryos, specifically at the post-otic level. The DR5 at the 3'flanking region of chick Hoxb1 is also capable of driving the same expression in chick embryos. We found that AmphiHox3 possesses a DR5-type RARE in its 5'flanking region, and this drives an expression pattern similar to the RARE element found in the 3' flanking region of AmphiHox1. Therefore, the location of these DR5-type RAREs is conserved in amphioxus and vertebrate Hox clusters. Our findings demonstrate that conserved RAREs mediate RA-dependent regulation of Hox genes in amphioxus and vertebrates, and in vertebrates this drives expression of Hox genes in both neural crest and neural tube. This suggests that Hox expression in vertebrate neural crest cells has evolved via the co-option of a pre-existing regulatory pathway that primitively regulated neural tube (and possibly epidermal) Hox expression.