We investigated whether the uptake of a specific antipsychotic agent, sulpiride, in Caco-2 cells is mediated by a carrier-mediated system. Caco-2 cell monolayers were cultured in plastic culture dishes and uptake and efflux studies were conducted. The determination of sulpiride was performed by HPLC. At 37 °C, sulpiride uptake in pH 6.0 was twice as much as in pH 7.4. At 4 °C, however, no significant difference was observed between pH 6.0 and 7.4. The uptake at 4 °C was markedly lower than that obtained at 37 °C. The subtraction of the uptake at 4 °C from the uptake at 37 °C indicated a saturable process, and the result of the Eadie–Hofstee plot analysis indicated that the uptake consists of two or more saturable components. The uptake was significantly inhibited by uncoupler, protonophore, amino acid modifying agent and proteinase. Sulpiride efflux was temperature-dependent and was significantly inhibited by uncoupler and amino acid modifying agent. These findings indicate that sulpiride uptake and efflux in Caco-2 cells are carrier-mediated. Furthermore, the uptake was significantly decreased by some substrates and inhibitors of peptide transporter, PEPT1, and organic cation transporters, OCTN1 and OCTN2, and was significantly increased by preloading with them. The uptake was also significantly increased by a typical substrate of P-glycoprotein. From these findings, we presumed that peptide transporter PEPT1 and organic cation transporters OCTN1 and OCTN2 are involved with this uptake. P-glycoprotein may also contribute to the efflux of sulpiride.