Conservation practices from the perspective of functional diversity (FD) and conservation prioritization need to account for the impacts of exotic species in freshwater ecosystems. This work firstly simulated the influence of exotic species on the values of FD in a schemed mechanistic model, then a practical case study of conservation prioritization was performed in the Min River, the largest river in southeastern China, to discuss whether including exotic species alters prioritization. The mechanistic model revealed that exotic species significantly altered the expected FD if the number of exotic species occupied 2% of the community. Joint Species Distribution Modeling indicated that the highest FD occurred in the west, northwest, and north upstreams of the Min River, the largest river in southeastern China. Values of FD in 64.69% of the basin were decreased after removing the exotic species from calculation. Conservation prioritization with the Zonation software proved that if firstly removing the habitats of exotic species during the process of prioritization, 62.75% of the highest prioritized areas were shifted, average species representation of the endemic species was improved, and mean conservation efficiency was increased by 7.53%. Existence of exotic species will significantly alter the metrics of biodiversity and the solution for conservation prioritization, and negatively weighting exotic species in the scope of conservation prioritization is suggested to better protect endemic species. This work advocates a thorough estimate of the impacts of exotic species on FD and conservation prioritization, providing complementary evidence for conservation biology and valuable implications for local freshwater fish conservation. This article is protected by copyright. All rights reserved.