Research Article published by SpringerThe stable isotopes of δ18O, δ2H, and 87Sr/86Sr and dissolved major ions were used to assess spatial and seasonal water chemistry variability, chemical weathering, and hydrological cycle in the Pangani River Basin (PRB), Tanzania. Water in PRB was NaHCO3 type dominated by carbonate weathering with moderate total dissolved solids. Major ions varied greatly, increasing from upstream to downstream. In some stations, content of fluoride and sodium was higher than the recommended drinking water standards. Natural and anthropogenic factors contributed to the lowering rate of chemical weathering; the rate was lower than most of tropical rivers. The rate of weathering was higher in Precambrian than volcanic rocks. 87Sr/86Sr was lower than global average whereas concentration of strontium was higher than global average with mean annual flux of 0.13 × 106 mol year−1. Evaporation and altitude effects have caused enrichment of δ18O and δ2H in dry season and downstream of the river. Higher d-excess value than global average suggests that most of the stations were supplied by recycled moisture. Rainfall and groundwater were the major sources of surface flowing water in PRB; nevertheless, glacier from Mt. Kilimanjaro has insignificant contribution to the surface water.We recommend measures to be taken to reduce the level of fluoride and sodium before domestic use.