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ABSTRACT The flavor quality of citrus fruits is largely determined by the sugar-acid ratio, but it remains uncertain how sugar- and/or acid-metabolizing enzymes regulate the sugar-acid ratio of navel oranges and further affect the fruit quality. In the present study, Robertson navel oranges (Citrus sinesis Osb.) were collected from six representative habitats in three eco-regions of Sichuan, China. The changes in the sugar-acid ratio and the activities of sucrose phosphate synthase (SPS), sucrose synthase (SS), cytosolic cio-aconitase (ACO), and isocitrate dehydrogenase (IDH) were examined in navel oranges during fruit development. The results indicated that the sugar-acid ratio of fruits in different eco-regions changed significantly from 150 days after full bloom. The SPS and cytosolic ACO fruit activities had minor changes among different ecoregions throughout the experimental periods, whereas the activities of SS and IDH changed significantly in fruits among three eco-regions. Furthermore, the sugar-acid ratio and the activities of SS in the synthetic direction and IDH were the highest in south subtropics and the lowest in north mid-subtropics, probably due to the effects of climate conditions and/or other relevant eco-factors. It demonstrated that SS in the synthetic direction and IDH were of greater importance in regulating the sugar-acid ratio of navel oranges in different eco-regions, which provided new insights into the factors that determine the flavor quality of navel oranges and valuable data for guiding relevant agricultural practices.