Metformin is frequently prescribed for the treatment of type 2 diabetes in humans. Recent detections of this drug in samples collected from racehorses in the United States and subsequent pharmacokinetic studies have demonstrated a prolonged terminal half-life in horses, suggesting slow redistribution or delayed elimination processes. In humans, studies have demonstrated that metformin partitions into red blood cells (RBCs) resulting in a prolonged residence time in the body. In the current study, it was hypothesized that intracellular sequestration of metformin may account for the prolonged terminal elimination phase observed in horses. Six Thoroughbred horses were administered a single oral dose of metformin (15 grams) and blood and urine samples collected. Metformin concentrations were determined in plasma, serum, whole blood, RBCs, and urine using liquid chromatography tandem mass spectrometry and non-compartmental pharmacokinetic analysis was conducted. An additional set of serum samples were collected for determination of the effect of hemolysis on serum metformin concentrations. Metformin reached peak concentrations in RBCs between 25 minutes and 2 hours, with RBC concentrations surpassing serum concentrations from approximately 24 hours onward.Serum concentrations exceeded RBC concentrations from 264 hours until termination of sample collection. The terminal half-life of metformin was 75.4 ± 32.2 hours and 49.1 ± 7.01 hours for serum and RBCs, respectively. Serum metformin concentrations did not appear to be altered by experimentally induced hemolysis. Urine concentrations varied greatly between horses and concentrations fluctuated unpredictably with some horses showing decreases followed by increases at later time points. Consistent with observations in humans, metformin demonstrates preferential partitioning into RBCs in horses. Red blood cells may function as a reservoir, gradually releasing metformin back into the circulation, thereby prolonging its detectability in serum.