The loosening is one of the main failure modes of bolted joints. Currently, researchers only focused on bolt loosening behavior under simple axial or transverse loads. Understanding the loosening failure mechanism of bolted joints under complex load is important for improving reliability. A multi-directional loading test device was designed, and bolt loosening experiments were conducted. The effect of load direction and amplitude on bolt loosening behavior were investigated. The results indicate that bolt loosening can be divided into two stages. Load direction and load amplitude are critical factors determining the loosening trend in the later stage. In addition, a refined finite element model of bolted joints was established to verify and explain the experimental results. The FEA results show that the relative sliding between internal and external threads and the stress redistribution on the threads are the main reasons for the bolt loosening. The smaller the angle between the load direction and the tangential direction of the contact surface, the relative sliding more likely to occur.