Aim: Antofloxacin(ANT) is a newly developed fluoroquinolone antibacterial in China. Pharmacokinetic study in healthy volunteers shows that 40%-45% of ANT administered is eliminated by kidney. The effect of human organic transporter 1 on renal tubular secretion of ANT was investigated in present study. Methods: HK-2 cell monolayer model was established and its integrity of the model was confirmed by transephitherial electric resistance (TEER) and the diffusion rate of fluorescein-labeled dextran (molecular mass 40 kDa). The activity of hOAT1 was evaluated by p-aminohippurate (PAH). Bi-directional transport of ANT solely or co-used with probenecid (PRO) were studied by using HK-2 cell monolayer model to determine whether hOAT1 affected renal tubular secretion of ANT. The concentrations of ANT collected from the transport assay were determined by high performance liquid chromatography and the transport parameters such as clearance (CLA-B and CLB-A) were calculated and compared when the ANT was used solely and co-used with PRO. Results: When cells was growing in 5-7 d, TEER became stable and the range was from 140 to 150 OM.cm^2. CLA-B and CLB-A of PAH and ANT were 0.192+/-0.027, 0.325+/-0.184 and 0.475+/-0.200, 0.636+/-0.060 of 60 min transport, respectively. While co-transported with PRO, CLA-B and CLB-A of PAH and ANT became 0.399+/-0.035, 0.171+/-0.017(p<0.05) and 0.772 +/- 0.120,0.422 +/- 0.032 respectively (p<0.05). Conclusion: The above results proved HK-2 cell monolayer model can be used to study the hOAT - drug interaction and hOAT1 affect the secretion of ANT from renal tubular basal side (B) into apical side (A).