A communication region defining method is a method for defining a defined communication region in operation of a toll collection system having only an on-board unit present within the defined communication region as a communication target for toll collection and includes: a transmitter arrangement step of arranging reference transmitters capable of transmitting radio waves at a plurality of positions on a boundary line of a defined communication region; a position location step of locating a position of each of the plurality of reference transmitters on the basis of a position location signal obtained by receiving, through predetermined position location antennas, radio waves transmitted by the plurality of reference transmitters; and a determination condition setting step of setting conditions for determining whether to set an on-board unit as a communication target for toll collection on the basis of position location results with respect to the plurality of reference transmitters.