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 results in increased translation, particularly for transcripts involved in carbon assimilation and nitrogen metabolism. OsNOP2-KO boosts grain yield ~28% per plot in the Nipponbare genetic background in normal condition and maintains increased yield traits under both heat treatment and saline soil conditions. More importantly, knockout of OsNOP2 in the rice varieties Longgeng31 and Xiushui134, as well as its orthologs in wheat and tomato, also increases the RNA m5C level to enhance yield, supporting functional conservation of OsNOP2's regulatory impacts. Together, our findings unveil an RNA m5C elevating mechanism by OsNOP2 that epigenetically governs carbon assimilation and nitrogen utilization efficiency in plant, providing a potential strategy for genetic improvement in multiple crops.