Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of neural crest Krox20-expressing cells reveals a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20 deficiency embryos. Genetic lineage tracing in Krox20−/− mutant mice shows that endothelial-derived cells are normal whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells using is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings unravel a critical role of Krox20 in arterial valve development and reveal that excess of neural crest cells may be associated to bicuspid aortic valve.