Myocellular creatine (Cr) uptake is predominantly governed by a sodium-dependent creatine transporter (CreaT) and plays a pivotal role in skeletal muscle energy metabolism. The CreaT belongs to a neurotransmitter transporter family that can be functionally regulated by protein tyrosine kinase induced tyrosine phosphorylation. The association between myocellular Cr and c-Src related tyrosine phosphorylation of the CreaT and the influence of oral Cr supplementation on this association were investigated during sepsis. Animals were randomized to receive standard rat chow or standard rat chow with oral Cr supplementation for four days followed by cecal ligation and puncture (CLP) or sham-operation. Fast-twitch gastrocnemius muscles were harvested 24 hours after operation. Myocellular free Cr levels were 70% higher after CLP. Western blotting of the immunoprecipitated CreaT with an anti-phospho-tyrosine or anti-phospho-c-Src (Y416) antibody revealed that tyrosine phosphorylation of the CreaT and tyrosine phosphorylated c-Src (Tyr-416) expression in the CreaT-c-Src (Y416) complex were significantly increased after CLP compared to sham-operation. These changes were observed in homogenates and plasma membrane fractions of gastrocnemius muscles. Although oral Cr supplementation increased myocellular free Cr levels equivalently in CLP and sham-operated animals, c-Src related tyrosine phosphorylation of the CreaT in homogenates and plasma membrane fractions of gastrocnemius muscles was, however, down-regulated in Cr-supplemented CLP animals as compared to Cr-supplemented sham-operated rats. During sepsis, increased myocellular free Cr levels are associated with enhanced tyrosine phosphorylation of the CreaT, which is likely induced by active c-Src. Oral Cr supplementation down-regulates c-Src related tyrosine phosphorylation of the CreaT. The data suggest that myocellular Cr homeostasis and CreaT activity are tightly regulated and closely related during sepsis.