To better understand the mechanisms regulating plant carotenoid metabolism in staple crop, we report the map‐based cloning and functional characterization of the Glycine max carotenoid cleavage dioxygenase 4 (GmCCD4) gene, which encodes a carotenoid cleavage dioxygenase enzyme involved in metabolizing carotenoids into volatile β‐ionone. Loss of GmCCD4 protein function in four Glycine max increased carotenoid content (gmicc) mutants resulted in yellow flowers due to excessive accumulation of carotenoids in flower petals. The carotenoid contents also increase three times in gmicc1 seeds. A genome‐wide association study indicated that the GmCCD4 locus was one major locus associated with carotenoid content in natural population. Further analysis indicated that the haplotype‐1 of GmCCD4 gene was positively associated with higher carotenoidlevels in soybean cultivars and accumulated more β‐carotene in engineered E. coli with ectopic expression of different GmCCD4 haplotypes. These observations uncovered that GmCCD4 was a negative regulator of carotenoid content in soybean, and its various haplotypes provide useful resources for future soybean breeding practice.