Pseudomonas are ubiquitous bacteria that can live under a wide range of environmental conditions; some species are successfully used in the fermentation industry. Pseudomonas plecoglossicida JUIM01 strain is an important industrial 2-ketogluconate (2KGA) producer. In Pseudomonas, kguT gene encodes a putative 2KGA transporter KguT, and there is no detailed research on the role of KguT. In this work, the role of KguT in P. plecoglossicida JUIM01 was investigated. The results showed that the KguT of P. plecoglossicida JUIM01 plays an essential role in the transport of 2KGA into cells; inactivation of KguT could prevent the assimilation of 2KGA as carbon source by the cells and had no negative effect on 2KGA biosynthesis. Under fermentation conditions, the kguT deletion strain had the same capability of 2KGA production with P. plecoglossicida JUIM01 and avoided the assimilation of the product 2KGA by cells after the depletion of glucose. These results can provide references for Pseudomonas study and fermentation production of 2KGA.