Land use changes are known to alter terrestrial silicon cycling and the export of dissolved silicon from soil to fluvial systems, but the impact of such changes on groundwater systems remain unclear. In order to identify the processes responsible for groundwater geochemistry and to assess the impact of agricultural processes, we examined multiple isotopic tracers (δ30Si, oxygen (δ18O) and hydrogen (δ2H) isotopes) in groundwater, soil porewater and surface water from two contrasted watersheds having the same gneissic lithology, one forested (Mule Hole) and one intensely cultivated (Berambadi) in the Kabini basin in South India. In the cultivated watershed, groundwater exhibits high Cl− and NO3− concentrations indicative of fertilizer inputs and solute enrichment from evapotranspiration due to multiple groundwater pumping/recharge cycles. The DSi concentration in groundwater is significantly higher in the cultivated watershed (980 ± 313 μM) than in the forested one (711 ± 154 μM), indicating more intense evapotranspiration due to irrigation cycles. The groundwater δ30Si values ranged from 0.6 ‰ to 3.4 ‰ and exhibit no significant differences between cultivated (1.2 ± 0.5 ‰) and forested (1.0 ± 0.2 ‰) watersheds, indicating limited impact of land use and land cover. Groundwater also shows no significant seasonal differences in DSi and δ30Si within watersheds, indicating a buffer to seasonal recharge during wet season. The δ30Si of a majority of groundwater samples fits a steady-state open flow through system, with an isotopic fractionation factor (30ε) between precipitating phase and groundwater ranging from −1.0 ‰ and − 2.0 ‰, consistent with precipitation of kaolinite-type clays, dominant in the study area. The steady-state flow through system in groundwater can be interpreted as a continuous DSi input from mineral weathering reactions with a dynamic equilibrium between Si supply and precipitation of secondary phases. We also observe, in both watersheds, similar DSi and δ30Si values in local surface water that includes small streams and a river (406 ± 194 μM, 1.6 ± 0.3 ‰) and in soil porewater (514 ± 119 μM, 1.6 ± 0.2 ‰). Compared to soil porewater, groundwater exhibits significantly lower δ30Si signatures and higher DSi, reflecting the contribution of an isotopically light silicon source, resulting from water-rock interaction during percolation through the unsaturated zone. We assign this steady input of DSi to the weathering of primary silicate minerals in the regolith, such as Na-plagioclase, biotite and chlorite, with formation of kaolinite and smectites type clays. A simple isotopic mass balance suggests that deep regolith weathering can contribute to almost half of the DSi in groundwater. We conclude that silicon cycling in soil porewaters, and surface waters are directly impacted by land use, while the isotopic composition of groundwater remains unaffected. Our results indicate that Si isotopic signatures of weathering, adsorption, and plant uptake occurring in the shallow soil and saprolite horizons are partly overprinted and homogenized by the regolith weathering in the deep critical zone, irrespective of land use and seasonality.