Bioactive gibberellins (GAs, diterpenes) are essential hormones in land plants, controlling many aspects of plant growth and developments. In flowering plants, 13-OH (low bioactivity; such as GA1) and 13-H GAs (high bioactivity; such as GA4) frequently coexist. However, the bona fide GA 13-hydroxylase and its physiological functions in Arabidopsis remain unknown. Here, we report that novel cytochrome P450 genes (CYP72A9 and its homologs) encode active GA 13-hydroxylases in Brassicaceae plants. CYP72A9-overexpressing plants exhibited semi-dwarfism, which was caused by significant reduction in GA4 levels. Biochemical assays revealed that recombinant CYP72A9 protein catalyzed the conversion from 13-H GAs to the corresponding 13-OH GAs. CYP72A9 was expressed predominantly in developing seeds in Arabidopsis. Freshly harvested seeds of cyp72a9 mutants germinated more quickly than wild-type, while long-term storage and stratification-treated seeds did not. The evolutionary origin of GA 13-oxidases from the CYP72A subfamily also was investigated and discussed here.