We explored in-gap states (IGSs) in perovskite oxide heterojunction films. We report that IGSs in these films play a crucial role in determining the formation and properties of interfacial two-dimensional electron gas (2DEG). We report that electron trapping by IGSs opposes charge transfer from the film to the interface. The IGS in films yielded insulating interfaces with polar discontinuity and explained low interface carrier density of conducting interfaces. An ion trapping model was proposed to explain the physics of the IGSs and some experimental findings, such as the unexpected formation of 2DEG at the initially insulating LaCrO3/SrTiO3 interface and the influence of substitution layers on 2DEG.