Spermine is the end-product in the polyamine biosynthetic pathway, and its excess accumulation induces neuroexcitatory responses and neurotoxicity. The purpose of this study was to elucidate the involvement of transport systems at the brain barriers in the clearance of spermine. In vivo rat spermine elimination from brain parenchyma across the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier (BCSFB) was assessed by intracerebral and intracerebroventricular administration techniques, respectively. To characterize spermine transport at the BCSFB, a transport study using rat choroid plexus was performed. After the intracerebral microinjection of [³H]spermine, no time-dependent decrease in [³H]spermine in the ipsilateral cerebrum was observed, suggesting the low contribution of the BBB to spermine clearance from the brain. In contrast, the [³H]spermine concentration in the CSF after intracerebroventricular administration was time-dependently decreased with an elimination rate constant of 0.352 min⁻¹, and the elimination clearance of [³H]spermine was 6.6-fold greater than that of [¹⁴C]D-mannitol, reflecting bulk flow of the CSF. This [³H]spermine elimination was attenuated by co-administration of unlabeled excess spermine, indicating carrier-mediated elimination of spermine from the CSF. [³H]Spermine transport into the choroid plexus was strongly inhibited by unlabeled spermine, other polyamines (spermidine and putrescine), and organic cation transporter substrates such as corticosterone and 1-methyl-4-phenylpyridinium. However, other substrates/inhibitors for organic cation transporters (decynium-22 and tetraethylammonium) had little effect. Consequently, our study indicates that transporting molecules at the BCSFB, distinct from typical organic cation transporters, are involved in spermine clearance from the CSF.