<p>Chilling injury during the booting stage of rice is a significant constraint on stable production in high-latitude and high-altitude areas. Despite its importance, the role of transcriptional and lipidomic alterations in rice anthers under cold conditions in conferring cold tolerance remains poorly understood. In this study, analyses of pollen fertility and lipid staining revealed that the cold-tolerant variety, LJ31, exhibited more intense pollen and lipid staining compared to the cold-sensitive variety, LD3. Transcriptome analysis identified lipid metabolism as pivotal determinant of the observed differences in cold tolerance between two genotypes, with 174 differentially expressed genes (DEGs) associated with lipid metabolic processes. Lipidomic profiling further demonstrated that LJ31 enhances the biosynthesis of digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) by upregulating enzymes such as MGD and SQD1. These changes contribute to improved membrane fluidity and stability, thereby enhancing cellular resilience to cold stress. Additionally, the accumulation of primary membrane phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TG), provides critical energy reserves essential for mitigating the adverse effects of cold stress. In contrast, the cold-sensitive variety, LD3, dexhibited a marked reductio in total lipid content, primarily driven by decreased production of phosphatidic acid (PA), suggesting inefficiencies in the conversion of diacylglycerol (DG) to PA. This metabolic disorder affects signaling and membrane synthesis. Furthermore, reductions in PC and SQDG levels in LD3 may undermine membrane integrity and functionality, resulting in diminished cellular stability under cold conditions. These DEGs-induced alterations in lipid metabolism form a regulatory network that is vitally important for further exploring the molecular mechanisms by which rice anthers cope with cold environments.</p>