[Objective] To analyze changes in ecosystem water regulation services before and after the implementation of ecological projects in the Three-River headwaters region, Qinghai Province, and to evaluate the ecological effect of these projects objectively.[Methods] This paper calculated the water regulation amount of forest and grassland ecosystems with rainfall storage capacity method.[Results] The water regulation services of forest and grassland ecosystems increased in fluctuations from 1997 to 2012, with the averaged value of 1.54×10¹⁰ m³/a within the region and 430.67 m³/hm² per unit area. The regulated amount of water in the later period(2005-2012) increased by 15.60% than that in the earlier period(1997-2004). Spatially, water regulation service capacity gradually decreased from southeast to northwest. Water regulation amount per unit area of different basins in descending order was Lantsang River basin > Yellow River basin > Yangtze River basin. Water regulation amount after the implementation of ecological projects was higher than before under both real climate conditions and mean climate conditions. In the earlier period, the water regulation service was mainly affected by regional climate change. While in the later period, it was overlaid the effects of ecological projects. In these two conditions, the linear trend of water regulation amount from 2005 to 2012 was 1.94×10⁹ m³/10 a and 4.64×10⁸ m³/10 a, respectively.[Conclusion] Implementation of the ecological projects had certain positive effects on improvement of water regulation service, with a contribution rate of about 23.98%.