Gear meshing stiffness is the key parameter to study the gear dynamic performance. However, the study on the calculation of gear meshing stiffness considering lubrication, especially mixed lubrication, is still insufficient. Based on the three-dimensional linear contact mixed elastohydrodynamic lubrication model and the contact stiffness calculation method of rough surface, a method for calculating the gear meshing stiffness under mixed lubrication is proposed in this paper. According to the proposed calculation method, the effects of speed, external load, and roughness amplitude on gear meshing stiffness are further explored. The method can take into account the real rough surface topography and lubrication in the meshing process, so it may be more advantageous than the conventional method to some extent.