Membrane transporters are widely expressed throughout the body, transporting nutrients, metabolites, toxins, and drugs across cellular membranes. In drug development, transporters are clinically important for drug absorption and disposition, and play significant roles in pharmacokinetics and treatment response. In addition, there is a growing interest in physiologic roles of transporters as polymorphisms in many drug transporters have been significantly associated with human diseases. Metformin is widely used as first-line treatment of type 2 diabetes. It exists primarily as a hydrophilic cation at physiological pHs, and therefore, membrane transporters are involved in its intestinal absorption, tissues distribution, and renal elimination. Multiple organic cation transporters play roles in the pharmacokinetics of metformin, and many of them play important roles in its pharmacological response, as mediators of metformin entry into target tissues. Here, we investigated transporters of metformin, with an emphasis on the pharmacological and biological roles of transporters in the intestine and liver. We characterized the human thiamine transporter (THTR-2; SLC19A3), which plays a major role in the intestinal absorption of thiamine (Vitamin B1). We found that human THTR-2 is capable of transporting other compounds including the drugs metformin and famotidine, and the neurotoxin MPP+. Other chemical agents including phenformin, chloroquine, verapamil, famotidine, amprolium, and pyrithiamine were inhibitors of hTHTR-2 mediated uptake of both thiamine and metformin. Interestingly, previous studies from our laboratory and others identified thiamine as an endogenous substrate for several organic cation transporters including OCT1, OCT2, and MATE1. In our second study, we focused on the endogenous role of OCT1, which is the major hepatic transporter for metformin. Through extensive experiments in Oct1 knockout mice, our data support the notion that hepatic thiamine deficiency is the underlying mechanism for the clinical phenotypes associated with reduced OCT1 function, particularly elevated LDL- cholesterol and total cholesterol levels. Our data suggest that reduced OCT1-mediated thiamine uptake in the liver leads to reduced levels of thiamine pyrophosphate, TPP, and a decreased activity of key TPP-dependent enzymes. As a result, there is a shift from glucose to fatty acid oxidation in the liver, which disrupts key metabolic flux pathways. In parallel, through analysis of published genomewide association studies, we show that reduced function OCT1 polymorphisms are associated with several metabolic traits in humans. Taken together, our studies suggest that changes in OCT1 activity modulate the disposition of thiamine and its active metabolite, TPP, which results in alterations in hepatic energy metabolism, ultimately affecting total body energy homeostasis. Finally, we developed a comparative structural model of OCT1 that discriminates ligands from non-ligands and is the model together with an in vitro high-throughput screening (HTS) assay to screen a drug library to explore the molecular properties of drugs interacting with OCT1. To extend the in silico methods in predicting endogenous metabolites that interact with OCT1, we conducted a virtual screen of 29,332 human metabolites against the structure-based model and an SAR model generated from the HTS data. In conclusion, our findings have important implications for understanding metformin absorption and disposition, and have expanded our understanding of the physiologic and pathophysiologic roles of membrane transporters.