The roller linear motion guide (RLMG) is a key transmission component in heavy-load mechanical systems, and its internal non-uniform load distribution and stiffness greatly affect the stability of mechanical systems. To determine the accurate non-uniform load distribution and stiffness of the RLMG, a new approach is proposed by considering the variations of the contact angle, roller-raceway contact state, and energy loss. The variations of the contact angle are calculated by considering the initial contact angle, preload, and block flexibility. The non-uniform load distribution of each roller is studied under various roller-raceway contact states. The energy loss is calculated according to the sliding friction between contact surfaces under the loading process. Then, a new stiffness matrix is developed by modifying the energy balance equations, which are iteratively solved using the artificial bee colony algorithm. The variations of the contact angle, non-uniform load distribution, energy loss, and stiffness with external load and preload are discussed. The proposed approach is compared with other models and published experimental results. The comparison results demonstrated that the proposed approach is more precise than the other models.