In some cloud forest systems, fog water intercepted by the canopy plays a significant role in ecohydrological processes, while its importance on groundwater systems remains poorly understood. In this study, a cloud forest system in the lower reaches of the Chishui River was selected as an example to explore the contribution of fog interception to the local groundwater system using isotope hydrology methods. From March 2021 to February 2022, samples of rainwater, fog water, and spring water in the Jinshagou catchment of the Chishui Cyathea Reserve were collected and analyzed for 2H and 18O isotopes. Using long time-series data, temporal variation rules and causes of isotopic composition in rainwater, fog interception, and groundwater were revealed, based on which an isotope mixing model was established to quantify fog interception’s proportion in groundwater. Results showed that δ2H, δ18O, and d-excess values of rainwater exhibited obvious seasonal fluctuations, indicating the seasonal alternation of water vapor sources. Compared to other southwestern regions, the rainwater of the study area was characterized by higher d-excess values especially during the dry season, indicating a more significant component of terrestrial recirculated water vapor source. Fog interception water was more enriched in heavier isotopes than rainwater, but they had a similar seasonal variation pattern, suggesting their similar water vapor source. Due to the relatively higher condensation temperature of fog, however, the d-excess of fog water was slightly lower than that of rainwater. No seasonal isotopic variation was observed in spring water, suggesting that groundwater has a long residence time and is a long-term mixture of atmospheric water recharge. The averaged isotopic values of groundwater were higher than the weighted mean of annual rainwater, proving the joint recharge from both rainwater and fog water. The isotope mixing model estimated that the proportion of fog water in the groundwater of the Chishui Cyathea Reserve was 25.5% ~ 28.1%, corresponding to a water input of 367 mm ~ 419 mm. Compared with previous studies, this study further considers the seasonal changes of water isotopes, thus reducing uncertainties in the quantitative results. In summary, fog interception has a significant water contribution to the groundwater system in the study area, and its ecohydrological effect is important for sustaining the local Cyathea population.