Radix astragali and moxifloxacin are always used together for active tuberculosis in China clinics. This study investigates the effects of astragaloside IV (AS-IV, the main components of Radix astragals) on the pharmacokinetics of moxifloxacin in rats. The pharmacokinetics of orally administered moxifloxacin (40 mg/kg) with or without AS-IV pretreatment (100 mg/kg/day for 7 days) was investigated. The plasma concentration of moxifloxacin was determined using LC-MS method, and the pharmacokinetics profiles were calculated and compared. Caco-2 cell transwell model was also used to investigate the effects of AS-IV on the transport pf moxifloxacin. The results showed that when the rats were pretreated with AS-IV, the maximum plasma concentration (C-max) of moxifloxacin decreased from 4.53 to 3.47 mu/mL (P < 0.05), and the area under the concentration-time curve from zero to infinity (AUC(0-inf)) also decreased from 21.17 to 16.06 mg-h/L (P < 0.05). The Caco-2 cell transwell experiments indicated that AS-IV could increase the efflux ratio of moxifloxacin from 2.54 to 3.58 through inducing the activity of P-gp. In conclusion, these results indicated that AS-IV could affect the pharmacokinetics of moxifloxacin, possibly by decreasing the systemic exposure of moxifloxacin by inducing the activity of P-gp.