Ryan Donaldson, who left the Heed for the U's last summer, provided the cross for Liam Hughes to head in after the break and then curled home a free-kick.
Gateshead struggled to turn possession into chances, until Jack Lester headed into an empty net to get one back.
Liam Hatch and James Curtis nodded wide in injury time but the U's held on.
It was only last season that Newcastle-born Donaldson scored eight times for Gateshead, but he consigned his former club to a 55th year outside of the Football League.
Cambridge, meanwhile, can sympathise with their opponents, having lost at this stage in 2008 and 2009 in their attempts to end their exile in non-league.
The experience of winning the FA Trophy at Wembley back in March would have no doubt helped the U's on the big occasion, but they had to end a 14-game unbeaten run by the Tynesiders and did so in ordinary time.
A Wembley crowd of over 20,000 saw the game, which was also shown live on BT Sports.