Increasing evidence has shown that nanocarriers have effects on several efflux drug transporters. To date, little is known about whether influx transporters are also modulated. Herein, we investigated the impact of amphiphilic polymer micelles on the uptake function of organic cation transporters (OCTs) and the influence on the pharmacokinetics and pharmacodynamics of metformin, a well-characterized substrate of OCTs. Five types of polymeric micelles (mPEG(2k)-PCL2k, mPEG(2k)-PCL3.5k, mPEG(2k)-PCL5k, mPEG(2k)-PCL7.5k, and mPEG(2k)-PCL10k) were prepared to evaluate the inhibition of hOCT1-3-overexpressing Madin-Darby canine kidney cells. The mPEG(2k)-PCLx micelles played an inhibitory role above the critical micelle concentration. The inhibitory potency could be ranked as mPEG(2k)-PCL2k > mPEG(2k)-PCL3.5k > mPEG(2k)-PCL5k > mPEG(2k)-PCL7.5k > mPEG(2k)-PCL10k, which negatively declined with the increase of molecular weight of the hydrophobic segment. The inhibitory effects of polymeric micelles on the hOCT1 isoform were the most pronounced, with the lowest IC50 values ranging from 0.106 to 0.280 mg/mL. The mPEG(2k)-PCL2k micelles distinctly increased the plasma concentration of metformin and significantly decreased V-ss by 35.6% (p < 0.05) after seven consecutive treatments in rats, which was interrelated with the restrained metformin distribution in the liver and kidney. The uptake inhibition of micelles on hepatic and renal rOcts also diminished the glucose-lowering effect of metformin and fasting insulin levels in the oral glucose tolerance test. Consistent with the inhibitory effects, the mRNA and protein levels of rOct1 and rOct2 were decreased in the liver, kidney, and small intestine. The present study demonstrated that mPEG(2k)-PCLx micelles could inhibit the transport function of OCTs, indicating a potential risk of drug-drug interactions during concomitant medication of nanomedicine with organic cationic drugs.