Proton transfer is classified into two mechanisms: the Grotthuss (proton-relay) and vehicle mechanisms. It has been well studied on gas-phase proton transfer by a proton relay involving multiple molecules. However, a vehicle mechanism in which a single molecule transports a proton has rarely been reported. Here, we have obtained clear evidence that the proton transfers efficiently between the two protonation sites in protonated p-aminobenzoic acid (PABA·H+) by a single ammonia molecule as a vehicle. The gaseous PABA·H+ ions were reacted with NH3 or ND3 under single-collision conditions in a cold ion trap, and the proton-transferred ions were identified by cryogenic ion mobility-mass spectrometry. A reaction intermediate PABA·H+·NH3 was also detected for the first time. The reaction pathway search calculations and ab initio molecular dynamics simulations supported the present experimental finding that intramolecular proton transfer occurs very efficiently by the vehicle mechanism.