Despite substantial drought conditions in the karst critical zone (KCZ), the KCZ landscapes are often covered with forest woody plants. However, it is not well understood how these plants balance water supply and demand to survive in such a water-limited environment. This study investigated the water uptake and transpiration relationships of four coexisting woody species in a subtropical karst forest ecosystem using measurements of microclimate, soil moisture, stable isotopes (δ 18O, δ 2H, and δ 13C), intrinsic water-use efficiency (WUE i), sap flow, and rooting depth. The focus was on identifying differences within- and between-species across soil- and rock-dominated habitats (SDH and RDH) during the rainy growing season (September 2017) and dry season (February 2018). Species across both habitats tended to have higher transpiration with lower WUE i during the rainy season and lower transpiration with higher WUE i during the dry season. Compared to those in the SDH, species in the RDH showed lower transpiration with higher WUE i in both seasons. The dominant water sources were soil water and rainwater for supporting rainy-season transpiration in the SDH and RDH, respectively, and groundwater was the main water source for supporting dry-season transpiration in both habitats. A clear ecohydrological niche differentiation was also revealed among species. Across both habitats, shallower-rooted species with higher soil-water uptake, compared to deeper-rooted species with higher groundwater uptake, showed higher transpiration and lower WUE i during the rainy season and vice versa during the dry season. This study provides integrated insights into how forest woody plants in the KCZ regulate transpiration and WUE i in response to drought stress through interactions with seasonal water sources in the environment.