Studying the factors influencing ecosystem regulation services in southwestern Zhejiang is of great significance for formulating reasonable pricing strategies for forest ecosystem regulation services and optimizing ecological security. This study constructed a theoretical framework for analyzing forest ecosystem regulation services and assessed the spatiotemporal evolution and influencing factors of forest ecosystem regulation services using InVEST model calculations and spatial autocorrelation analysis. The results showed that all ecosystem services of forests in the study improved from 2000 to 2019, with the exception of soil conservation. The water conservation function increased significantly from 2000 to 2019, with an overall increase of 3.53%. The biodiversity conservation function in 2019 also increased significantly, with an average increase of 2.16% compared with 2000. The synergies mainly occurred between water source regulation and soil conservation, soil conservation and biodiversity, and forest recreation and carbon storage. Forest Reserve was precipitation, canopy closure, elevation, and soil texture, and their driving forces differed at different time scales. The trade-offs mainly occurred between soil conservation and forest recreation, forest recreation and biodiversity, and carbon storage and biodiversity. The research results provide a reference for achieving ecological protection and high-quality development in the southwestern region of Zhejiang.