Background: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. Results: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. Conclusions: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning. Developmental Dynamics 241:627–637, 2012. © 2012 Wiley Periodicals Inc.