In order to clarify the deformation mechanism of Inconel 718 (IN718) alloy at the grain scale during tensile deformation, the deformation behaviors of IN718 alloy were investigated at 650 °C using an in situ electron backscatter diffraction (EBSD) tensile testing method. The evolution of grain morphology, crystallographic orientation, activated slip systems, grain boundaries evolution, and strain-induced misorientation were systematically analyzed during the tensile test. The results showed that the grains were elongated along the tensile direction, and the grain boundaries also became significantly curved. Meanwhile, the EBSD studies illustrated that the changes in local misorientation within individual grains were non-uniform and generally began at the grain boundaries. The low-angle grain boundaries (LAGBs) were first formed near the high-angle grain boundaries (HAGBs) and gradually expanded into the interior of the grains. The activation of the slip system and the Schmid factor were characterized and calculated based on the slip traces on the deformed grain surface. The evolution of local strain within the grains was evidenced by a kernel average misorientation (KAM) map. Finally, the plastic deformation mechanism at the grain scale was discussed in detail based on our experimental results.