The involvement of OsKASI in FA synthesis is found to play a critical role in root development of rice. The root system plays important roles in plant nutrient and water acquisition. However, mechanisms of root development and molecular regulation in rice are still poorly understood. Here, we characterized a rice (Oryza sativa L.) mutant with shortened roots due to a defect in cell elongation. Map-based cloning revealed that the mutation occurred in a putative 3-oxoacyl-synthase, an ortholog of β-ketoacyl-[acyl carrier protein] synthase I (KASI) in Arabidopsis, thus designated as OsKASI. OsKASI was found to be ubiquitously expressed in various tissues throughout the plant and OsKASI protein was localized in the plastid. In addition, OsKASI deficiency resulted in reduced fertility and a remarkable change in fatty acid (FA) composition and contents in roots and seeds. Our results demonstrate that involvement of OsKASI in FA synthesis is required for root development in rice.