RNA 5-methylcytidine (m5C) has been identified as a key epi-transcriptomic modification of mRNAs involved in regulating multiple post-transcriptional processes. Here, we found that knockout of the RNA m5C demethylase, OsNOP2, results in elevated m5C levels and positively influences numerous agronomic traits in rice. After verifying OsNOP2 RNA m5C demethylase function in vitro and in planta, we found that enhanced m5C levels arising from OsNOP2 knockout result in increased protein translation, particularly for genes involved in carbon assimilation and nitrogen metabolism. Phenotypic analysis of OsNOP2-KO rice lines showed that grain yields per plot increased ~28% compared to wild-type Nipponbare under field conditions, and knockout lines maintain this increased yield phenotype under both heat treatment and high saline soil conditions. Additionally, OsNOP2 knockout confers similar effects in elite rice varieties, Longgeng31 and Xiushui134, while knockout of NOP2 orthologs in wheat and tomato also leads to increased RNA m5C levels and enhanced yield, supporting its functional conservation between monocots and dicots. This study thus illustrates an epigenetic regulatory role of OsNOP2 in controlling carbon and nitrogen metabolism in rice, and suggests that increasing m5C levels through NOP2 knockout could serve as a potentially effective strategy for genetic improvement of crops.