A semiconductor device includes active regions, including a first active region and a second active region, extending in a first horizontal direction, an isolation region defining the active regions, a gate structure disposed on the isolation region and extending in a second horizontal direction to intersect the active region, and separation structures penetrating through the gate structure and disposed on the isolation region between the first active region and the second active region. The separation structures include a first separation structure extending into the isolation region, and a second separation structure disposed on the first separation structure and penetrating through at least a portion of the first separation structure, and a width of a lower region of the second separation structure in the second horizontal direction is less than a width of an upper region of the first separation structure in the second horizontal direction.