Intrinsic water-use efficiency (iWUE) is a key physiological trait; however, the spatiotemporal variation in iWUE and which factors affect iWUE in the tropics and subtropics are poorly known. We determined the temporal (1920-2010) and spatial patterns of iWUE using leaf carbon-isotopic composition (δ¹³C) of 1,811 herbarium specimens and associated these patterns with environmental factors across biomes in southern China. We found that iWUE increased by 15.7 μmol mol⁻¹ in the entire studied area from 1920 to 2010. The iWUE declined from west to east in southern China, with higher values concentrated in the southwest. Rising CO₂ concentrations ([CO₂]), N deposition, increases in mean annual temperature (MAT) and vapor pressure deficit (VPD), and changes in temperature seasonality (TS) contributed to the spatiotemporal changes in iWUE. Our results confirm that, in addition to a continuous increase in iWUE has, the rate of change of iWUE decreased in southern China in the later years. Multiple atmospheric factors jointly determine the changes in both iWUE and the rate of change of iWUE in the studied regions.