The Tajik Basin is now located to the west of the Pamirs, but it formerly connected with the Tarim Basin, occupied by a large epicontinental sea (proto-Paratethys Sea) once extended from Europe to Central Asia. However, the northward indentation and collision of the Pamirs into the Tian Shan separated the Tajik and Tarim Basins, creating manifold implications and consequences. Consequently, timing of the final seawater retreat from the Tajik Basin is important for understanding the evolution of the proto-Paratethys Sea, the effect of the northward indentation of the Pamirs on the sea regression, and the influence of seawater retreat on the Cenozoic aridification in the Tajik Basin. Here we present a multidisciplinary study of Upper Paleogene strata from the central Tajik Basin. Our results demonstrate that the final seawater retreat from the center of the Tajik Basin occurred at 38.6 Ma, which is earlier than a previous age of 37.4 Ma from the northeastern Tajik Basin. Such a late Eocene sea retreat was quasi-simultaneous with that in the Tarim Basin. This final marine regression was mainly a consequence of the northward indentation and growth of the Pamirs, which was an intracontinental response to the India-Asia collision. Our new detrital zircon ages indicate that the provenance of Upper Eocene-lower Oligocene strata was from the Pamirs rather than Tian Shan, implying that the reactivation of the Tian Shan was later than the early Oligocene. Our paleomagnetic data indicate a 30 degrees counterclockwise rotation of the central Tajik Basin that is much bigger than the previous estimate of 17 degrees, and we propose that such a counterclockwise rotation mostly occurred after the late Miocene. Finally, different from the previous reported aridfication at 39 Ma or 37 Ma, our evidence demonstrates that drought intensified since the start of the Oligocene at 34 Ma, being related to reduced water vapor transport to Central Asia, caused by global coolinginduced sea-level drop, the retreating Paratethys Sea, and weakened sea surface evaporation.