Abstract The aleurone in cereal grains is an outer cell layer enriched with multiple nutrients important for human health. Enhancing the thickness of the aleurone layer through breeding could improve the nutritional value of rice. In this study, we characterized OsABCB24, a member of the ABCB transporter gene subfamily in rice, and its role in regulating aleurone development. Expression profiling revealed that OsABCB24 is predominantly expressed in seedling leaves and developing caryopsis, particularly in aleurone layer cells during grain filling. Subcellular localization analyses via protoplast transfection and immunogold labeling demonstrated that OsABCB24 is localized to the chloroplast. Knockout of OsABCB24 significantly increased the thickness of the aleurone cells and elevated the concentrations of minerals such as phosphorus, potassium, zinc, magnesium, and copper in brown rice. Knockout of OsABCB24 also decreased the concentrations of free and conjugated indole-3-acetic acid (IAA) in developing caryopsis and increased the leaf angle by influencing cell proliferation and elongation on the adaxial side of the lamina joint at the seedling stage. Leaf angle was less sensitive to exogenous IAA in osabcb24 mutants than in the wild type. Taken together, these findings suggest that OsABCB24 is a negative regulator of aleurone cell expansion possibly by modulating auxin homeostasis. OsABCB24 is a promising genetic target for breeding rice with increased aleurone thickness and nutrient concentrations without yield penalty.