Objectives: Polymyxins are a last-line therapy to treat MDR Gram-negative bacterial infections. Nephrotoxicity is the dose-limiting factor for polymyxins and recent studies demonstrated significant accumulation of polymyxins in renal tubular cells. However, little is known about the mechanism of polymyxin uptake into these cells. Oligopeptide transporter 2 (PEPT2) is a solute carrier transporter (SLC) expressed at the apical membrane of renal proximal tubular cells and facilitates drug reabsorption in the kidney. In this study, we examined the role of PEPT2 in polymyxin uptake into renal tubular cells. Methods: We investigated the inhibitory effects of colistin and polymyxin B on the substrate uptake mediated through 15 essential SLCs in overexpressing HEK293 cells. The inhibitory potency of both polymyxins on PEPT2-mediated substrate uptake was measured. Fluorescence imaging was employed to investigate PEPT2-mediated uptake of the polymyxin fluorescent probe MIPS-9541 and a transport assay was conducted with MIPS-9541 and [3H]polymyxin B1. Results: Colistin and polymyxin B potently inhibited PEPT2-mediated [3H]glycyl-sarcosine uptake (IC50 11.4 ± 3.1 and 18.3 ± 4.2 μM, respectively). In contrast, they had no or only mild inhibitory effects on the transport activity of the other 14 SLCs evaluated. MIPS-9541 potently inhibited PEPT2-mediated [3H]glycyl-sarcosine uptake (IC50 15.9 μM) and is also a substrate of PEPT2 (Km 74.9 μM). [3H]polymyxin B1 was also significantly taken up by PEPT2-expressing cells (Km 87.3 μM). Conclusions: Our study provides the first evidence of PEPT2-mediated uptake of polymyxins and contributes to a better understanding of the accumulation of polymyxins in renal tubular cells.