We investigated the moisture origin and contribution of different water sources to surface runoff entering the headwaters of the Heihe River basin on the basis of NECP/NCAR (National Centers for Environmental Prediction/National Center for Atmospheric Research) re-analysis data and variations in the stable hydrogen and oxygen isotope ratios (δD and δ¹⁸O) of precipitation, spring, river, and melt water. The similar seasonality in precipitation δ¹⁸O at different sites reveals the same moisture origin for water entering the headwaters of the Heihe River basin. The similarity in the seasonality of δ¹⁸O and d-excess for precipitation at Yeniugou and Urumchi, which showed more positive δ¹⁸O and lower d-excess values in summer and more negative δ¹⁸O and higher d-excess values in winter, indicates a dominant effect of westerly air masses in summer and the integrated influence of westerly and polar air masses in winter. Higher d-excess values throughout the year for Yeniugou suggest that in arid inland areas of northwestern China, water is intensively recycled. Temporal changes in δ¹⁸O, δD, and d-excess reveal distinct contributions of different bodies of water to surface runoff. For example, there were similar trends for δD, δ¹⁸O, and d-excess of precipitation and river water from June to September, similar δ¹⁸O trends for river and spring water from December to February, and similar trends for precipitation and runoff volumes. However, there were significant differences in δ¹⁸O between melt water and river water in September. Our results show that the recharge of surface runoff by precipitation occurred mainly from June to mid-September, whereas the supply of surface runoff in winter was from base flow (as spring water), mostly with a lower runoff amount.