Abstract: Ecosystem service has become an effective management agency to promote the harmony and unity of ecosystems and human society. The ecosystem service functions of food supply, water conservation, climate regulation and habitat quality of the Huaihe River Eco-economic Zone (HRECZ) were evaluated during the past 20 years by using market value method, water balance equation method, InVEST model and HSI model, respectively. The spatial-temporal variation and related reasons of four ecosystem services were analyzed. The results revealed that:The spatial pattern of ecosystem services in HRECZ exhibited great spatial heterogeneity; the distribution of food supply function had strong regional characteristics with respect to different provinces, and the good part of food supply service was shifting from northwest to southwest with noticeable temporal changes; For water conservation function, temporal variation was also obvious with increasing at early stage and then decreasing, and spatially, the maximum water source conservation amount of per unit grid area was 1 439.15 mm, whereas the minimum was only -407.65 mm; The cumulative carbon sequestration in HRECZ from 1995 to 2015 was 203.7 Tg, showing certain carbon sequestration function, and the sinking rate was fast at the early stage while becoming slowly progressively; The proportion of areas with good habitat quality in the HRECZ decreased, and the average score of habitat quality reached the maximum in 2005 and 2010, however, after then, it showed a downward trend. In the future, HRECZ should balance the spatial distribution of ecosystem services and the land to be developed. Cross-regional ecological protection and management cooperation should be carried with the guide of river basin governance. Suggestions are made available for the comprehensive development of HRECZ, and the protection, regulation and management of its ecosystem regulation.