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Groundwater plays a dominant role in the eco-environmental protection of arid and semi-arid regions. Understanding the sources and mechanisms of groundwater recharge, the interactions between groundwater and surface water and the hydrogeochemical evolution and transport processes of groundwater in the Longdong Loess Basin, Northwest China, is of importance for water resources management in this ecologically sensitive area. In this study, 71 groundwater samples (mainly distributed at the Dongzhi Tableland and along the Malian River) and 8 surface water samples from the Malian River were collected, and analysis of the aquifer system and hydrological conditions, together with hydrogeochemical and isotopic techniques were used to investigate groundwater sources, residence time and their associated recharge processes. Results show that the middle and lower reaches of the Malian River receive water mainly from groundwater discharge on both sides of valley, while the source of the Malian River mainly comes from local precipitation. Groundwater of the Dongzhi Tableland is of a HCO3–Ca–Na type with low salinity. The reverse hydrogeochemical simulation suggests that the dissolution of carbonate minerals and cation exchange between Ca2+, Mg2+ and Na+ are the main water-rock interactions in the groundwater system of the Dongzhi Tableland. The δ18O (from –11.70‰ to –8.52‰) and δ2H (from –86.15‰ to –65.75‰) values of groundwater are lower than the annual weighted average value of precipitation but closer to summer-autumn precipitation and soil water in the unsaturated zone, suggesting that possible recharge comes from the summer-autumn monsoonal heavy precipitation in the recent past (≤220 a). The corrected 14C ages of groundwater range from 3,000 to 25,000 a old, indicating that groundwater was mainly from precipitation during the humid and cold Late Pleistocene and Holocene periods. Groundwater flows deeper from the groundwater table and from the center to the east, south and west of the Dongzhi Tableland with estimated migration rate of 1.29–1.43 m/a. The oldest groundwater in the Quaternary Loess Aquifer in the Dongzhi Tableland is approximately 32,000 a old with poor renewability. Based on the δ18O temperature indicator of groundwater, we speculate that temperature of the Last Glacial Maximum in the Longdong Loess Basin was 2.4℃–6.0℃ colder than the present. The results could provide us the valuable information on groundwater recharge and evolution under thick loess layer, which would be significative for the scientific water resources management in semi-arid regions.