Mariners closer Brandon Morrow (1-2) retired the first two batters in the ninth before walking pinch-hitter Billy Butler on four pitches. DeJesus hit Morrow's next pitch, a fastball in, just over the right-field fence and into the Royals' bullpen.
"I kind of fist-pumped a little too early," DeJesus said. "I saw Ichiro [Suzuki] go back to the wall and I was like, 'oh geez, its not going to leave.' Then it went out and I was like, all right."
DeJesus' game-ending homer was the first by a Royal since Alberto Castillo on April 19, 2005, against Cleveland.
"That was my first ever walk-off hit of any kind at any level," DeJesus said. "Once I got in there the guys were punching and banging the heck out of me. It was fun seeing all those guys around home plate. It was pretty cool."
Six innings earlier, Grudzielanek had the second of his two hits to become the 18th active player with 2,000 hits. He was the 251st to reach the milestone in big league history.
"That's just icing on the cake with David's two-run shot in the bottom of the ninth," Grudzielanek said. "You dream about that as a kid, hitting a home run to win the game. Coming on a night that's very personal and very special to me."
Manager Trey Hillman said he predicted DeJesus' home run.
"That's one I actually called I said to Dave, 'We've got two outs. How about Billy drawing a walk and then you going boom?'" Hillman said. "Fortunately, it happened. That's good for us.
Morrow has blown saves in his past two opportunities, giving up four runs on three home runs.
"That was a fastball down the middle that I have gotten away with plenty of times before," Morrow said. "The walk really hurt."
Mariners manager Jim Riggleman agreed.
"The walk to Butler was crucial. He didn't bite on a couple of breaking balls that were very close. Once he gets to 2-0, he can now be very selective. He never got a strike and he took ball four. That was key because we were facing the bottom part of the lineup with a great arm. Now we're facing the top of the lineup. DeJesus got him. There's not much more you can say about it."
Left-hander Horacio Ramirez (1-0), who was with the Mariners last year, pitched 2 1-3 perfect innings of relief for the win.
Mariners starter Jarrod Washburn held the Royals to three runs -- one earned -- and seven singles over six innings. In his past seven starts, Washburn is 3-1 with a 2.58 ERA. He has allowed two earned runs or less in six of his past seven starts to lower his ERA from 6.56 to 4.83.
Royals starter Gil Meche was staked to a 3-0 lead and held his former team to four singles through five innings. But he ran into trouble in the sixth.
Suzuki led off with a single and with one out, Raul Ibanez crushed Meche's 2-0 pitch for his 11th home run. Ibanez had been 1-for-15 against the Royals this season before the home run.
"I would have been really upset if we hadn't won this game after Grud's 2,000th hit," Meche said. "I'm just glad we won this game for him and the team. I made some mistakes and before I could blink four runs scored and they had the lead."
Meche gave up four runs and 10 hits in 6 2/3 innings.
Tug Hulett, the son of former major leaguer Tim Hulett, made his big league debut as the Mariners designated hitter and singled in the fifth for his first hit. ... Royals DH Jose Guillen returned to the Royals lineup and went 1-for-4 after missing the game Friday night due to a stiff neck.