All-nitride Josephson junctions are being actively explored for applications in superconducting quantum chips because of their unique advantages including their antioxidant chemical stability and high crystal quality. However, the theoretical research on their microstructure mechanism that determines transport properties is still absent, especially on the defects. In this paper, we apply the first principles and non-equilibrium Green’s function to calculate the electrical transport characteristics of the yellow preset model. It is first revealed that the N-vacancy defects play a crucial role in determining the conductivity of the NbN-based Josephson junctions, and demonstrate the importance for the uniformity of vacancy distribution. It is found that the uniform number of vacancies can effectively increase the conductance of Josephson junction, but the position distribution of vacancies has little effect on the conductance. The work clarifies the effect of the N-vacancy defects on the conductivity of the NbN-based Josephson junctions, which offers useful guidance for understanding the microscope mechanism of the NbN-based Josephson junction, thus showing a great prospect in the improvement of the yield of superconducting quantum chips in the future.