Blonanserin (BNS), a new atypical antipsychotic drug with dopamine D2 and serotonin 5-HT2A antagonistic properties, is efficacious for use in patients with schizophrenia, and has been launched in Japan and South Korea. Several clinical trials suggest that BNS is noninferior to risperidone (RIS) for the primary endpoint (PANSS total score), and BNS has fewer reports than RIS of hyperprolactinemia (a major adverse effect of antipsychotics). BNS is a liposoluble compound and displays good brain distribution properties in preclinical studies, but its distribution mechanism has not been clarified. By contrast, RIS and its active metabolite, paliperidone (PAL), are known to be substrates of P-glycoprotein (P-gp/MDR1), an efflux transporter expressed in various tissues. Importantly, P-gp expressed in cerebral vascular endothelial cells limits entry of drugs (e.g. antipsychotics) into the central nervous system (CNS). In this study, using in vitro and in vivo approaches, we evaluated whether BNS, like RIS and PAL, is a P-gp substrate. In the in vitro study, affinity for P-gp at efficacious concentrations of BNS, RIS and PAL (around Cmax in clinical dosing) was investigated by transcellular transport system across LLC-PK1 and LLC-PK1 expressing human MDR1. In the in vivo study, the exposure of these drugs in the brain and plasma (B/P ratio) was measured in mdr1a/1b knockout (KO) and wild-type (WT) mice (ip, 1 mg/kg). The results of our in vitro study indicated that P-gp actively transports RIS and PAL, but not BNS, as substrates (net flux ratio: 3.4, 5.2 and 1.0, respectively). Furthermore, in the in vivo study, BNS gave comparable B/P ratios in KO and WT mice, whereas, B/P ratios for RIS and PAL differed markedly between these animals (8.2 and 17.1-fold for RIS and PAL, respectively). These results suggest that P-gp is likely to influence the brain distribution of RIS and PAL, but not that of BNS.