In vertebrates, paralogous Hox genes play diverse biological roles. We examined the interchangeability of Hoxa1 and Hoxb1 in mouse development by swapping their protein-coding regions. Remarkably, the mice expressing the Hox-B1 protein from the Hoxa1 locus, and vice versa, are essentially normal. We noted, nonetheless, a specific facial nerve hypomorphism in hemizygous Hoxb1(A1/-) mice and decreased viability in homozygous Hoxa1(B1/B1) embryos. Further, we established a mouse line in which we have inserted the 107 bp Hoxb1 autoregulatory enhancer into the Hoxa1 promoter. Strikingly, the newly generated autoregulatory Hoxa1 gene can deliver the functionality of both paralogs in these mice, providing normal viability as well as proper facial nerve formation even in the Hoxb1 mutant background. This study affirms that subfunctionalization of the transcriptional regulatory elements has a principal role in the diversification of paralogous Hox genes. Moreover, we show that the ancestral vertebrate Hox1 gene can still be experimentally reconstructed.