Nicotine binds to nicotinic acetylcholine receptors on dopaminergic terminals to evoke dopamine (DA) release. The clearance of released DA occurs rapidly through reuptake into nerve terminals through the DA transporter (DAT). However, whether nicotine modulates DAT function in vivo is still not well understood. In the present study, we determined the effect of nicotine on DA clearance using in vivo amperometric recording in the striatum of urethane-anesthetized rats. Stable DA release was evoked by electrical stimulation of the medial forebrain bundle (MFB). Subsequently, nicotine or saline was administered with MFB stimulation at 10-min intervals for 60 min. Kinetic analysis revealed that nicotine decreased the amplitude of DA overflow and the maximal DA clearance rate (Vmax) in response to stimulation of 96 pulses at 80 Hz. Surprisingly, nicotine enhanced the maximal DA clearance rate (Vmax) by stimulation of 768 pulses at 80 Hz. Furthermore, we found that this paradoxical effect of nicotine on Vmax depended on the stimulation pattern. These results suggest that nicotine may exert its addictive role by dynamically modulating DAT function in vivo.