The regression of the Neotethys Ocean from the southwestern Tianshan Mountains remains debatable because of notable sedimentary discontinuities and lack of fossil evidence. This study aimed to explain this problem by applying comprehensive carbonate UPb dating to newly recognised pyroclastic rock layers in the Tuoyun Basin, southwestern Tianshan Mountains. These rocks, referred to as peperite in the early literature, are not like typical pyroclastic rocks; rather, they are formed by the mixing of magma with wet sediment. Field observations have revealed that the pyroclastic rock layers are overlying Cenozoic pillow basalts and then covered by the later columnar jointing basalts, recording a transformation from marine to terrestrial environment. The dolomite-dominated mineralogy and major element analysis indicate that the pyroclastic rock is dolomitic in composition. The sedimentary origin is supported by the δ18OSMOW values (23.7‰ to 36‰) and δ13C values (−6.48‰ to −7.08‰) of the bulk-rock pyroclastic rock and the δ34S values (−20.79‰ to 23.86‰) of native sulfur. However, the relatively depleted initial ratios of 87Sr/86Sr (0.7048–0.7065) and εNd(t) values (−0.52 to +3.10) suggest the possibility of isotopic exchange with the conjugate basalts. For carbonate from Tuoyun pyroclastic rock, UPb isotope dating using laser ablation multi-collector inductively coupled plasma mass spectrometry (LA–MC–ICP–MS) produced well-defined Lutetian ages of 46.1 ± 0.5–41.7 ± 0.6 Ma. This indicates that the Tuoyun region was in a marine environment during the Lutetian epoch and then transformed into a continental environment after the marine regression. In this study, we successfully applied the UPb technique by LA–MC–ICP–MS to constrain the timing of ocean–continent transformation through carbonate UPb dating. These findings explain the regression of the Neotethys Ocean from the southwestern Tianshan Mountains and offer novel insights into the geological evolution history of this region.