In this paper, a novel criterion is developed for preventing the self-loosening of preloaded threaded cap screws under cyclic transverse loading. For a known cyclic excitation, the system parameters are used in the formulation of a closed form solution for the minimum fastener preload required for preventing self-loosening. The effect of several key variables is investigated; this includes bearing and thread friction coefficients, cap screw grip length, thread pitch, material, and cyclic amplitude of the transverse excitation. An experimental setup and test procedure are established. Comparison between the experimental and analytical clamp load variation results shows that the proposed criterion can accurately predict the requirements for preventing self-loosening.