Skeletal muscle serves as the body's major glutamine repository, and releases glutamine at enhanced rates during diabetes, but whether all muscles are equally affected is unknown. System N(m) activity mediates most trans-sarcolemmal glutamine movement, and although two System N (SN) isoforms have been identified (SN1/sodium-coupled neutral amino acid transporter or System N and A transporters [SNAT]-3; and SN2/SNAT5), their expression in skeletal muscle remains controversial. Here, the impact of Type I diabetes on glutamine uptake and System N transporter expression were examined in fast- and slow-twitch skeletal muscle from spontaneously diabetic (BB/Wor-DP) rats. Net glutamine uptake in fast-twitch fibers was decreased 75%-95%, but enhanced more than 2-fold in slow-twitch muscle from diabetic animals relative to nondiabetic controls. Both SNAT3 and SNAT5 mRNA were expressed in both muscle fiber types and their abundance was unaffected by diabetes. This represents the first report of differential fiber-specific effects of diabetes on skeletal muscle glutamine transport and the co-expression of distinct System N transporters in skeletal muscle.