We previously showed that anthraquinones (including rhein, emodin, aloe-emodin, chrysophanol and physcion) were inhibitors of human organic anion transporter 1 (hOAT1) and hOAT3, causing transporter-mediated drug–drug interactions in rats. In this study, the time-dependent inhibition (TDI) of hOAT1 and hOAT3 by anthraquinones was investigated. Madin–Darby canine kidney (MDCK)-hOAT1, HEK293-hOAT3 and their parental cells were used. Preincubation with chrysophanol or physcion for 30 min significantly increased the inhibition of hOAT1, but preincubation with rhein, emodin, aloe-emodin or probenecid had no effect on hOAT1 activity. By contrast, preincubation of hOAT3 with emodin, aloe-emodin, chrysophanol or physcion for 30 min significantly increased its inhibition, but preincubation with rhein or probenecid had no effect on activity. As the incubating time lengthened, from 0 to 60 min, both the inhibition of hOAT1 by chrysophanol and physcion and the inhibition of hOAT3 by emodin, aloe-emodin, chrysophanol and physcion were observed to increase in a time-dependent manner. In conclusion, our results suggest that some anthraquinones contribute to the TDI of hOAT1 and hOAT3. An inhibition study without the preincubation procedure may underestimate the inhibitory potential of anthraquinones against hOAT1 and hOAT3. The underlying mechanisms of TDI of hOAT1 and hOAT3 need to be further investigated.