Salt stress severely limits rice productivity, yet the genetic basis of natural variation and the underlying mechanism remain poorly understood. Here, through genome-wide association analysis and fine-mapping, we identify a major quantitative trait locus (QTL) gene, Tolerance to Salt 1 (TOLS1), that negatively regulates salt tolerance in rice. Overexpression of TOLS1 increases salt sensitivity, whereas loss of function enhances salt tolerance. TOLS1 interacts with the small guanosine triphosphatase (GTPase) OsRacB and enhances its GTPase activity, thereby attenuating salt-induced cytosolic Ca2+ elevation. We further identify a natural coding variation, SNP9 (+1018 A/G), in TOLS1 that contributes to functional divergence, with the TOLS1A allele exerting a stronger negative effect on salt tolerance than TOLS1G. Collectively, these findings reveal that natural variation in TOLS1 modulates OsRacB-Ca2+ signaling and thereby contributes to salt tolerance divergence in rice. This study provides mechanistic insight into how TOLS1 allelic variation regulates salt signaling and offers a potential target for molecular design breeding.