Polydactyly is a common malformation and can be an isolated anomaly or part of a pleiotropic syndrome. The elucidation of the mutated genes that cause polydactyly provides insight into limb development pathways. The extra-toes spotting (Xs) mouse phenotype manifests anterior polydactyly, predominantly in the forelimbs, with ventral hypopigmenation. The mapping of Xs(J) to chromosome 7 was confirmed, and the interval was narrowed to 322 kb using intersubspecific crosses. Two mutations were identified in eukaryotic translation initiation factor 3 subunit C (Eif3c). An Eif3c c.907C>T mutation (p.Arg303X) was identified in Xs(J), and a c.1702_1758del mutation (p.Leu568_Leu586del) was identified in extra-toes spotting-like (Xsl), an allele of Xs(J). The effect of the Xs(J) mutation on the SHH/GLI3 pathway was analyzed by in situ hybridization analysis, and we show that Xs mouse embryos have ectopic Shh and Ptch1 expression in the anterior limb. In addition, anterior limb buds show aberrant Gli3 processing, consistent with perturbed SHH/GLI3 signaling. Based on the occurrence of Eif3c mutations in 2 Xs lines and haploinsufficiency of the Xs(J) allele, we conclude that the Xs phenotype is caused by a mutation in Eif3c, a component of the translation initiation complex, and that the phenotype is associated with aberrant SHH/GLI3 signaling.