Disclosed are a separator for lipid bilayer membrane formation capable of forming a lipid bilayer membrane with excellent properties, wherein the separator for lipid bilayer membrane formation has sufficient mechanical strength and can be easily manufactured in a large scale by using a general-purpose machine without need of using an expensive machine, and a method of producing the separator. The separator for lipid bilayer membrane formation includes a thin film having one or more through holes and made of a resin capable of being wet-etched, and reinforcing layers covering both surfaces of the thin film and made of a resin capable of being wet-etched. The reinforcing layers cover the whole area of the thin film, except for the through holes and the peripheries thereof, and each through hole has a tapered cross-sectional shape.