A three-dimensional image reconstruction method of reconstructing a three-dimensional structural model of membrane proteins, the membrane proteins extending through a lipid membrane in a direction vertical to the membrane surface of the lipid membrane and rotated at random relative to an axis vertical to the membrane surface of the lipid membrane and wherein adjacent membrane proteins are spaced apart by random spacing, said method comprising the steps of: obtaining a first transmission electron microscope image of a sample taken by illuminating an electron beam on the sample containing the membrane proteins present within the lipid membrane from a direction tilted relative to a line normal to a membrane surface of the lipid membrane; obtaining a second transmission electron microscope image of the sample, after the first transmission electron microscope image is obtained, by illuminating the electron beam on the sample perpendicularly to the membrane surface of the lipid membrane, wherein a defocus value for taking the second transmission electron microscope image is greater than a defocus value for taking the first transmission electron microscope image, wherein the dose of said electron beam on the sample assumed when said second transmission electron microscope image is taken is greater than the dose of the electron beam on the sample assumed when said first transmission electron microscope image is taken; identifying orientations of the membrane proteins in the first transmission electron microscope image on a basis of a rotational angle relative to an axis perpendicular to the lipid membrane of the corresponding membrane proteins in the second transmission electron microscope image; and constructing a three-dimensional structure of the membrane proteins by extracting a plurality of particle images of the membrane proteins from the first transmission electron microscope image without performing extraction of particle images from the second transmission electron microscope image, classifying the extracted images according to the orientations of the membrane proteins identified in the step of identifying orientations of the membrane proteins, and averaging the classified particle images extracted from the first transmission electron microscope image. obtaining a first transmission electron microscope image of a sample taken by illuminating an electron beam on the sample containing the membrane proteins present within the lipid membrane from a direction tilted relative to a line normal to a membrane surface of the lipid membrane; obtaining a second transmission electron microscope image of the sample, after the first transmission electron microscope image is obtained, by illuminating the electron beam on the sample perpendicularly to the membrane surface of the lipid membrane, wherein a defocus value for taking the second transmission electron microscope image is greater than a defocus value for taking the first transmission electron microscope image, wherein the dose of said electron beam on the sample assumed when said second transmission electron microscope image is taken is greater than the dose of the electron beam on the sample assumed when said first transmission electron microscope image is taken; identifying orientations of the membrane proteins in the first transmission electron microscope image on a basis of a rotational angle relative to an axis perpendicular to the lipid membrane of the corresponding membrane proteins in the second transmission electron microscope image; and constructing a three-dimensional structure of the membrane proteins by extracting a plurality of particle images of the membrane proteins from the first transmission electron microscope image without performing extraction of particle images from the second transmission electron microscope image, classifying the extracted images according to the orientations of the membrane proteins identified in the step of identifying orientations of the membrane proteins, and averaging the classified particle images extracted from the first transmission electron microscope image.