<p>The fractionation mechanism and of sulfate sulfur isotopes such as polyhalite in Quaternary salt lakes and their indicative significance is deficient remain to be further investigated. The Kunteyi Salt Lake (KSL) in the Qaidam Basin (QB) contains representative Quaternary polyhalite, providing an opportunity to address the lack of geochemical evidence for its salt formation and evolutionary process. We reported δ³⁴S values from 15 polyhalite, 13 gypsum, 2 hexahydrite, and 1 bloedite samples taken from a sediment core in KSL. The δ³⁴S values ranged 13.4‰–26.7‰, with an average 19.8‰, showing significantly positive characteristics. The average δ³⁴S value of evaporite-layer minerals (polyhalite, hexahydrite, bloedite) is 19.3‰, slightly lower than that of gypsum from clastic layers (20.4‰). Combined with the existing age frame of the core and the related mineralogy, it showed that the replenishment of deep brine with high δ34S profoundly affected the isotope composition of KSL, in which bacterial sulfate reduction (BSR) and sulfate precipitation could play a role. Additionally, δ34S variations correlate with glacial-interglacial climate cycles in KSL, suggesting that sulfur isotopes in salt lake systems are sensitive to climate and environmental changes and hold potential as the index for paleoenvironmental studies. High and increasing δ34S values indicate the desalination process during the Marine Isotope Stage 5 (MIS5) and MIS7, while low and decreasing values indicate the concentration process during MIS6 and MIS8. This phenomenon inevitably coupled the process of the gradual separation between KSL and Qaidam paleolake until it dried up. This is the first study on the fractionation mechanisms and tracing significance of δ34S from the Quaternary salt lakes polyhalite, highlighting its great potential for determining salt-forming sources and paleoenvironments.</p>