Provided is a semiconductor device, including gate structures on a substrate, the gate structures extending parallel to a first direction and being spaced apart from each other by a separation trench interposed therebetween, each of the gate structures including insulating patterns stacked on the substrate and a gate electrode interposed therebetween; vertical pillars connected to the substrate through the gate structures; an insulating spacer in the separation trench covering a sidewall of each of the gate structures; and a diffusion barrier structure between the gate electrode and the insulating spacer.