The spatial resolution of pinhole single photon emission computed tomography （SPECT） imaging is usually estimated according to Anger’s empirical formula, which could result in a large inaccuracy in experimental measurement. We present a more accurate formula based on the analysis of physical processes of pinhole imaging. The effectiveness of the proposed formula was evaluated by accurate Monte Carlo simulations and experiments of the phantom imaging. The images were reconstructed using three\|dimensional iterative OSEM algorithm. Then the effects of the voxel size, the geometrical mapping of the projection matrix and the ratio of detector size to imaging object size, on the spatial resolution were discussed. The experiment results show that the spatial resolution estimated by the proposed formula is about 10% smaller than the experimental value while the estimation by the Anger’s formula is about 60% larger than the experimental value. Therefore, our proposed formula is more accurate than Anger’s formula for predicting the spatial resolution of pinhole SPECT imaging, and it can be utilized for design and use of pinhole SPECT system.