A three-dimensional semiconductor device includes a first substrate; a plurality of first transistors on the first substrate; a second substrate on the plurality of first transistors; a plurality of second transistors on the second substrate; and an interconnection portion electrically connecting the plurality of first transistors and the plurality of second transistors. Each of the plurality of first transistors includes a first gate insulating film on the first substrate and having a first hydrogen content. Each of the plurality of second transistors includes a second gate insulating film on the second substrate and having a second hydrogen content. The second hydrogen content is greater than the first hydrogen content.