P-glycoprotein (P-gp) is an energy-dependent transporter that contributes to the efflux of a wide range of xenobiotics at the blood–brain barrier playing a role in drug-resistance or therapy failure. In this study, we evaluated [ 123 I]-4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl)piperidine ([ 123 I]-FMIP) as a novel single photon emission computed tomography (SPECT) tracer for imaging P-gp at the brain in vivo.The tissue distribution and brain uptake as well as the metabolic profile of [ 123 I]-FMIP in wild-type and mdr1a (−/−) mice after pretreatment with physiological saline or cyclosporin A (CsA) (50 mg/kg) was investigated. The influence of increasing doses CsA on brain uptake of [ 123 I]-FMIP was explored. μSPECT images of mice brain after injection of 11.1 MBq [ 123 I]-FMIP were obtained for different treatment strategies thereby using the Milabs U-SPECT-II.Modulation of P-gp with CsA (50 mg/kg) as well as mdr1a gene depletion resulted in significant increase in cerebral uptake of [ 123 I]-FMIP with only minor effect on blood activity. [ 123 I]-FMIP is relative stable in vivo with >80% intact [ 123 I]-FMIP in brain at 60 min p.i. in the different treatment regiments. A dose-dependent sigmoidal increase in brain uptake of [ 123 I]-FMIP with increasing doses of CsA was observed. In vivo region of interest-based SPECT measurements correlated well with the observations of the biodistribution studies.These findings indicate that [ 123 I]-FMIP can be applied to assess the efficacy of newly developed P-gp modulators. It is also suggested that [ 123 I]-FMIP is a promising SPECT tracer for imaging P-gp at the blood-brain barrier.