Micro-defects in an energetic material is an important factor for the formation of “hot spots” and successive explosive detonation. However, an understanding of the micro-mechanism of forming “hot spots” is limited and the development and application of energetic materials are hindered due to the less knowledge of micro-defects inside the materials. In order to understand the characteristics of micro-defects and explore the basic mechanism of forming “hot spots” caused by defects, the effects of molecular vacancy defect on the geometrical structure, electronic structure and vibration characteristics of Hexogeon (RDX) energetic materials are studied using the first-principle method, and the basic formation mechanism of initial “hot spot” is discussed. The effects of molecular vacancy defect on the RDX geometrical structure, electronic band structure, electronic density of states and frontier molecular orbitals are analyzed using the periodic model, while the influences of molecular vacancy defect on the vibration characteristics of RDX systems are calculated using the cluster model. Infrared vibration spectra and vibration characteristics of the internal molecules at the same vibration frequency for the perfect and defective RDX systems are obtained. It is found that vacancy defect makes the N–N bond near the defect long, and the molecular structure loose; some degenerate energy levels in the conduction band present separation and the electronic density of states decreases; the bottom of the conduction band and the top of the valence band contributed by N-2p and O-2p orbitals shift to the Fermi surface, which reduces the energy band gap and increases the activity of system. At the same time, the calculations of the frontier molecular orbitals and the infrared vibration spectra show that the molecular defect makes the charge distributions of highest occupied moleculer orbital concentrated mainly in the molecule near the defect, and the C–H and N–N bond energies decrease. For the defective system, some molecules around vacancy have large vibration amplitude towards the vacancy direction. This will be likely to cause hole to collapse and realize the conversion of energy. These characteristics indicate that the presence of molecular vacancy defect causes the energy band gap to decrease, the structures of the molecules near the defect become loose, the charge distribution increases and the reaction activity augments. When the defective system is loaded by external energy, the molecules near the defect are expected to be unstable. The C–H or N–N bonds in those molecules are more prone to rupture to cause chemical reaction and release of energy, which is expected to be responsible for the forming of “hot spot”. These results provide some basic micro-information about revealing the formation mechanism of “hot spots” caused by molecular vacancy defects