Short-chain carboxylic acids and their derivatives are widely utilized in all aspects of our daily life. Given their specific functional groups, these molecules are also utilized in fine chemical synthesis. The traditional petroleum-based carboxylate production methods are restricted by petrol shortage and environmental pollution. Renowned for their more sustainable processes than traditional methods, biotechnological methods are preferred alternatives and have attracted increasing attention. However, the industrial application of biotechnological methods is currently limited by low factors: low productivity and low yield. Therefore, understanding the regulation of carboxylate accumulation will greatly enhance the industrial biotechnological production of short-chain carboxylate acids. The carboxylate transporter plays a crucial role in transmembrane uptake and secretion of carboxylate; therefore, regulating these transporters is of high academic and application relevance. This review concentrates on the physiological roles, regulation mechanisms, and harnessing strategies of Jen and AcpA orthologs in fungi, which provide potential clues for the biotechnological production of short-chain carboxylic acids with high efficiency.