ABSTRACT The water uptake depth of trees affects their overall water use and the water cycle of forest ecosystems. However, empirical data on the depth from which water is drawn are lacking for many moist forests. In this study, we estimated the water uptake depths of 24 coexisting tree species in a moist cool-temperate mixed forest in Japan using stable oxygen isotopes. Our goal was to clarify the factors influencing water uptake depth including season, species, slope position, and tree height under moist conditions. Water uptake depth was determined during three periods in which temperatures differed but soil moisture levels remained similar. Season was shown as one of the factors affecting water uptake depth, which was shallowest in August, when temperatures reached maximum values, and deepest during May, when temperatures were lowest. This seasonal variation is likely to be related to physiological processes such as increasing transpiration which leads to water uptake shifting to the water-rich shallow soil layer and/or activation of fine root production at the soil surface, where temperatures are high. Water uptake depth also varied with tree species, suggesting the occurrence of belowground resource segregation among moist forest tree species. Slope position and tree height did not affect water uptake depth. This study highlights the importance of considering temperature-driven physiological processes when examining water uptake depth in moist forests, which could have implications for predicting the responses of these ecosystems to climate change.