AbstractMicrobes play an important role in biotransformation and biosynthesis of biofuels, natural products, and polymers. Therefore, microbial manufacturing has been widely used in medicine, industry, and agriculture. However, common strategies including enzyme engineering, pathway optimization, and host engineering are generally inadequate to obtain an efficient microbial production system. Transporter engineering provides an alternative strategy to promote the transmembrane transfer of substrates, intermediates, and final products in microbial cells and thus enhances production by alleviating feedback inhibition and cytotoxicity caused by final products. According to the current studies in transport engineering, native transporters usually have low expression and poor transportation ability, resulting in inefficient transport processes and microbial production. In this review, current approaches for transporter mining, characterization, and verification are comprehensively summarized. Practical approaches to enhance the transport system in engineered cells, such as balancing transporter overexpression and cell growth, and evolution of native transporters are discussed. Furthermore, the applications of transporter engineering in microbial manufacturing, including enhancement of substrate utilization, concentration of metabolic flux to the target pathway, and acceleration of efflux and recovery of products, demonstrate its outstanding advantages and promising prospects.