The objective of this study was to investigate molecular and physiological changes in response to long‐term insulin glargine treatment in the skeletal muscle of OLETF rats. Male Otsuka Long‐Evans Tokushima Fatty (OLETF) and Long‐Evans Tokushima Otsuka (LETO) rats aged 24 weeks were randomly allocated to either treatment with insulin for 24 weeks or no treatment, resulting in three groups. Insulin glargine treatment in OLETF rats (OLETF‐G) for 24 weeks resulted in changes in blood glucose levels in intraperitoneal glucose tolerance tests compared with age‐matched, untreated OLETF rats (OLETF‐C), and the area under the curve was significantly decreased for OLETF‐G rats compared with OLETF‐C rats (P < 0.05). The protein levels of MHC isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of myosin heavy chain type I and II fibers were lower and higher, respectively, in OLETF‐G compared with OLETF‐C rats. Activation of myokines (IL‐6, IL‐15, FNDC5, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF‐G compared with OLETF‐C rats ( P < 0.05). MyoD and myogenin levels were decreased, while IGF‐I and GLUT4 levels were increased, in the skeletal muscle of OLETF‐G rats ( P < 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of AMPK, SIRT1, and PGC‐1α. Together, our results suggested that changes in the distribution of fiber types by insulin glargine could result in downregulation of myokines and muscle regulatory proteins. The effects were likely associated with activation of the AMPK/SIRT1/PGC‐1α signaling pathway. Changes in these proteins may at least partly explain the effect of insulin in skeletal muscle of diabetes mellitus.