Climate change and anthropogenic factors have significantly impacted hydrological regimes, especially in semiarid regions. Using the trend-free pre-whitening Mann-Kendall (TFPW-MK) test and linear regression, trends of precipitation, temperature, runoff, and water balances from different spatiotemporal scales were analysed during the past sixty years in the Upper Yongding River Basin (UYRB). Based on the Choudhury-Yang equation, the contributions of climate change and human activities to runoff variation were further quantified in five mountainous catchments, and the actual effects of climate change and human activities on the water balance in the whole UYRB were also discussed. The results suggest that the UYRB has become much warmer and drier. The lower annual precipitation and intensity in summer were directly attributed to runoff reductions. Intra-annual scale analyses of meteorological, hydrological and biological responses helped the comprehensive interpretation of the runoff trends. Human activities, including agricultural irrigation, were the main causes for runoff reduction, especially in the middle and lower reaches, and the contribution of human activities increased gradually in recent decades. The water balance changed drastically temporally, with the precipitation being converted less to runoff and more to evaporated fractions, which was also due to the second order effects of climate change and vegetation improvement. It is suggested that low water-consumption plant species and the related water-saving techniques in agriculture and afforestation should be introduced for the sustainable development in the similar regions.