Chilling stress severely threatens rice productivity. While multiple signaling pathways in chilling tolerance have been elucidated, the role of the Mediator complex in this process remains poorly understood. Here, we identify COG5, a major quantitative trait locus (QTL) gene encoding the Mediator complex subunit Med21, as a positive regulator of chilling tolerance in rice. A promoter variation in COG5 leads to differential expression between japonica and indica. OsDREB6 preferentially binds to and activates COG5jap rather than COG5ind, resulting in higher expression in japonica. COG5 interacts with OsbHLH002 and enhances its transcriptional activation activity to promote downstream cold-responsive gene expression. Evolutionary analysis reveals that COG5jap originated from Chinese wild rice and underwent significant positive selection during japonica domestication. Collectively, our findings reveal that natural variation in COG5 modulates the cold-responsive transcriptional network via Mediator-transcription factor interactions, providing valuable molecular insights and genetic resources for molecular design breeding aimed at improving rice chilling tolerance.