Rice (Oryza sativa L.), a thermophilic crop, is highly sensitive to cold stress, particularly during the seedling stage. Developing cold-tolerant rice varieties is a possible strategy to mitigate yield losses caused by low temperatures. However, few genes for cold tolerance have been identified. In this study, we identified OsALA4 (Aminophospholipid ATPase 4), encoding a plasma membrane-localized P4-ATPase, from a chromosomal segment substitution line (CSSL-K2832-2) harboring cold-tolerance QTL qLTS5 (Low Temperature Sensitive 5). Genetic and subcellular localization analyses revealed that OsALA4 regulates cold tolerance by maintaining plasma membrane fluidity and cellular homeostasis. Physiological assessments showed that OsALA4 reduces malondialdehyde (MDA), electrolyte leakage, reactive oxygen species (ROS), and cell death under cold stress. Promoter activity assays indicated that stronger OsALA4 expression in Nipponbare (OsALA4^Nip) correlated with enhanced cold tolerance. Further experiments demonstrated that SNP sites within the promoter regions (−1500 bp to −700 bp) of OsALA4^Nip and OsALA4⁹³¹¹ influenced their activity. This study highlights OsALA4 as a valuable genetic target for breeding cold tolerant rice.