The Three Gorges Reservoir area is the largest strategic reserve of fresh water resources in China, and its ecological location is extremely important in the Yangtze River region. Therefore, it is very necessary and crucial to build the protection forest system with water conservation as the main function in the Three Gorges Reservoir area. Masson pine forest is the most important forest type in the protection forest system of the Three Gorges Reservoir area. However, the eco-hydrological effects of natural secondary forest of Masson pine with more complex structure was not studied enough in the past, which led to a limitation to the complete understanding and the accurate evaluation of the water conservation service of the forests of the Three Gorges Reservoir area. Therefore, the hydrological effects of all functional layers (forest canopy layer, undergrowth herbaceous layer, humus layer and soil layer) of the natural secondary forests of Masson pine were systematically studied at Tieshanping of Chongqing, based on the field positioning observation and laboratory measurement. The results showed that:(1) the throughfall, stemflow and interception of Masson pine forests accounted for 84.66%, 0.26% and 15.07% of total precipitation (1972.39 mm) in 2010 and 2011, respectively, and all presented a significantly positive relation (P< 0.01) with the rainfall depth of individual rainfall events. The stemflow began to appear only when rainfall was more than 5 mm. The rainfall interception increased gradually with rising rainfall depth to its maximum value of about 6 mm. (2) The aboveground biomass of the undergrowth herbaceous was 1.32 t/hm². The water amount absorbed by the herbaceous increased logarithmically with soaking time (P<0.01) until to its maximum value of 0.61 mm. (3) The humus layer amounted to 10.74 t/hm². The ratios of the maximum water-holding capacity of the un-decomposed humus, half-decomposed humus, and mixture of un-decomposed and half-decomposed humus were 183.76%, 206.31% and 197.62%, respectively. The maximum water-holding capacity of both the un-decomposed and half-decomposed humus layer was 1.44 mm. (4) The saturated water-holding capacity of the soil layer (0-80 cm) was 334.75 mm, in which the non-capillary water-holding capacity was 49.08 mm (14.66%). In summary, all functional layers of the natural secondary forests of Masson pine in the studied site present an obvious service of rainfall interception and water storage, and especially the service of the soil layer is the highest. The result of this study can provide an important basis for the simulation and evaluation of the water conservation service of the forests in the Three Gorges Reservoir area.