The nature and timing of post-collisional crustal thickening and its link to surface uplift in the eastern Lhasa block of the southern Tibetan plateau remain controversial. Here we report on Cenozoic magmatism in the Wuyu area of the eastern Lhasa block. The Eocene (ca. 46 Ma) trachyandesites and trachydacites show slight fractionation of rare earth elements (REE), slightly negative Eu and Sr anomalies, and relatively homogeneous Sr-Nd and zircon Hf isotopes (87Sr/86Sr(i) = 0.7050–0.7063, εNd(t) = −0.92 to −0.03, εHf(t) = +2.6 to +4.8). Previous studies have suggested Neo-Tethys oceanic slab break-off at 50–45 Ma; thus, the Wuyu Eocene magmatism could represent a magmatic response to this slab break-off and originate from relatively juvenile Lhasa crust. The Miocene (ca. 15–12 Ma) dacites and rhyolites have adakitic affinities, e.g., high Sr (average 588 ppm), Sr/Y (29–136), and La/Yb (30–76) values, low Y (4–12 ppm) and Yb (0.4–0.9 ppm) contents, and variable Sr-Nd and zircon Hf isotopes (87Sr/86Sr(i) = 0.7064–0.7142, εNd(t) = −11.7 to −3.7, εHf(t) = −3.2 to +4.5). Their more enriched Sr-Nd-Hf isotopes relative to the Eocene lavas indicate that they should be derived from mixed Lhasa lower crust comprising juvenile crust, ultrapotassic rocks, and probably Indian lower crust-derived rocks. This study has also revealed the transformation from Eocene juvenile and thin crust with a thickness of <40 km to Miocene mixed and thickened crust with a thickness of >50 km. Combined with published tectonic data, we suggest that both lithospheric shortening and magma underplating contributed to eastern Lhasa block post-collisional crustal thickening. Given the spatial-temporal distribution of eastern Lhasa block magmatism and regional geology, we invoke a post-collisional tectonic model of steep subduction of the Indian plate and subsequent westward-propagating plate break-off beneath the eastern Lhasa block, which caused the surface uplift.