Protein lysine lactylation is a new post-translational modification (PTM) prevalently found in fungi and mammalian cells that directly stimulates gene transcription and regulates the glycolytic flux. However, lysine lactylation sites and regulations remain largely unexplored, especially in cereal crops. Herein, we report the first global lactylome profile in rice, which effectively identified 638 lysine lactylation sites across 342 proteins in rice grains. Functional annotations demonstrated that lysine lactylation was enriched in proteins associated with central carbon metabolism and protein biosynthesis. We also observed that proteins serving as nutrition reservoirs in rice grains were frequently targeted by lactylation. Homology analyses indicated that lactylation was conserved on both histone and nonhistone proteins across plants, human cells, and fungi. In addition to lactylation, additional types of acylations could co-occur in many proteins at identical lysine residues, indicating potential cross-talks between these modifications. Our study provided a comprehensive profile of protein lysine lactylation in cereal crop grains.