The BURP gene family encodes the BURP domain protein, a type of plant-specific protein that plays an important role in plant development, metabolism and stress resistance. Salt stress is one of the major stresses faced by rice. However, there is a lack of systematic understanding of the BURP family involved in the regulation of salt stress in rice, especially for the gene OsBURP12. Our study used japonica rice variety Zhonghua 11 (ZH11), as the wild type (WT), and its OsBURP12 knockout lines to fill the gap of the understanding. NaCl solution was used for salt stress treatment. Related physiological and biochemical analyses were carried out to investigate the regulatory mechanism of OsBURP12 on salt tolerance in rice seedlings. The results showed that the survival rate, seedling height, root length and fresh weight of the knockout lines were significantly higher than those of the WT under salt stress, indicating that the salt tolerance of the knockout lines was significantly better than that of the WT. Under salt stress, the knockout lines had significantly higher antioxidant enzyme activity, lower accumulation of ROS (reactive oxygen species) and higher expression of genes related to ROS-scavenging than the WT. In addition, the knockout lines showed significantly lower PG activity and Na+ content, but significantly higher pectin content, K+ and Ca2+ content than the WT. Furthermore, the knockout lines had significantly higher ABA and ACC levels than the WT. Quantitative PCR analysis showed that the expression levels of genes related to ABA and ethylene synthesis and signaling were significantly higher in the knockout lines than in the WT. The results suggested that the knockout of OsBURP12 can improve ROS-scavenging ability, affect PG activity, regulate Na+ uptake, and mediate the synthesis and metabolism of ABA and ACC in response to salt stress for a tolerance in rice. Our study laid the foundation for further analysis of the function of BURP family genes in plants, and provided a valuable genetic resource for studying the mechanism of salt tolerance in rice.