Three-dimensional printing can bring huge advantages when fabricating difficult structures and components in industry. This technology is also used commonly for printing threaded fasteners in the engineering field. The main disadvantage of threated fasteners is self-loosening. In this study, an International Organization for Standardization (ISO) standard M12 × 1.75 bolt and nut were printed by using a 3D printer and three types of 3D printing materials: acrylonitrile butadiene styrene (ABS-2), poly lactic acid (PLA), and glass. Following this, a test system for self-loosening of the 3D-printed bolts under cyclical temperature variations was constructed, and self-loosening of the bolted joints was observed by measuring the bolt preload using a load cell and the rotation displacement of the nut using a dial indicator according to temperature changes (from 10 °C up to 40 °C and 80 °C). The experimental results show that the ABS-2 bolt has good performance in terms of self-loosening phenomena under cyclical temperature changes, while the PLA bolt has poor performance in low temperature changes. The glass bolt indicated the lowest performance in high temperature changes.