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Rural areas are the main reserves of biodiversity resources and are also important distribution areas for China' s natural re⁃ serves. Opportunity cost, as the main cost of the construction of natural reserves, is a potential driving factor in the spatial distribution of natural reserves. This study analyzed the decision-making mechanism of natural reserves from the perspective of county-level govern⁃ ments, using county-level panel data from 2005 to 2018 to empirically analyze the impact of opportunity cost in rural areas on the distri⁃ bution of natural reserves, and discussed the impact according to the location and level of natural reserves. On this basis, this study fur⁃ ther explored the impact of opportunity cost on the individual scale of natural reserves. The results showed that: ① In general, opportu⁃ nity cost was an important factor in determining the distribution of natural reserves. There was a significant negative relationship be⁃ tween the opportunity cost and natural reserve areas. Each 1% increase in the opportunity cost would reduce the natural reserve areas by 0.679%. ② In the eastern and central regions, opportunity cost had a significant negative impact on the natural reserve areas, while in the western and northeastern regions, opportunity cost had no significant impact on the natural reserve areas. ③ Opportunity cost had a significant negative impact on the areas of local-level natural reserves, while having no significant impact on the areas of national-lev⁃ el natural reserves. ④ As the opportunity cost increased, the size of a single natural reserve had a tendency to shrink, which could alle⁃ viate the pressure of the opportunity cost. However, the habitat of the reserve faced the risk of fragmentation, which was not conducive to biodiversity conservation. We suggest that in the process of further optimizing the layout of natural reserves, China should fully con⁃ sider the impact of opportunity cost in rural areas on the establishment and management of natural reserves, improve the multi-function⁃ al protected area system with national parks as the main body, strictly approve the adjustment of the scope and functional areas of natu⁃ ral reserves at all levels, include objects with high ecological protection value and rich biodiversity in the protection system, and estab⁃ lish a diversified and market-based ecological compensation mechanism for natural reserves to alleviate the conflict between the con⁃ struction of natural reserves and the development of surrounding communities, so as to achieve the dual goals of biodiversity conserva⁃ tion and economic development in rural areas. [ABSTRACT FROM AUTHOR]