In plants, polyglutamylated folate forms account for a significant proportion of the total folate pool. Polyglutamylated folate forms are produced by the enzyme folylpolyglutamate synthetase (FPGS). The FPGS enzyme is encoded by two genes in rice, Os03g02030 and Os10g35940. Os03g02030 represents the major expressed form in developing seed. To determine the function of this FPGS gene in rice, a T-DNA knockout line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed filling. LC-MS/MS-based metabolite profiling revealed that the abundance of mono- and polyglutamylated folate forms was significantly decreased in seeds of the knockout line. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knockout plant, whereas the folate deglutamating enzyme ?-glutamyl hydrolase mRNA level was reduced. Our study has uncovered a novel role for folate polyglutamylation during rice seed development and a potential feedback mechanism to maintain folate abundance.