[Objective] The national park system pilot is an important component of China’s ecological civilization development. Investigating its impact on newly established enterprises in the region is of great significance for achieving synergy between ecological conservation and economic development. [Methods] This study took the Wuyishan National Park pilot as its research subject. Based on the registration data of newly established enterprises in counties across Fujian Province (including counties, county-level cities, and municipal districts under prefecture-level cities) from 2000 to 2023, this study employed event study methodology, difference-in-differences, and machine learning models to reveal the impact of the national park system pilot on regional newly established enterprises and its underlying mechanisms. [Results] (1) The national park system pilot policy had a positive impact on the number of newly established enterprises at the county level. (2) Heterogeneity analysis showed that the national park system pilot had a stronger effect on the number of large enterprises than on medium, small, and micro enterprises, and a greater effect on private enterprises than on state-owned enterprises, Hong Kong-funded, Macao-funded, and Taiwan-funded enterprises, and foreign-funded enterprises. (3) Further analysis showed that the national park system pilot promoted industrial structure upgrading at the county level by attracting newly established enterprises in sectors such as culture, sports, and entertainment, accommodation and food services, and wholesale and retail trade. [Conclusion] The national park system pilot promotes the growth of the number of regional newly established enterprises, optimizes the regional industrial structure, and contributes to high-quality regional economic development. Local governments should formulate differentiated policies for enterprise development, support the development of green industries, and thereby achieve win-win coordination between ecological conservation and economic development.