The basketball legend had wanted Beckham to appear on his new reality show Shaq Vs, which will see him taking on fellow sports stars at their own game.
Writing on his Twitter blog, he said: "Dear David Beckham. I know you have heard about my Shaq Vs show, anyway you will never score a goal on me. I challenge you, lil' man.
"David Beckham, I know you hear me. Don't be scared, don't make me call you out. Don't make me Tweet to two million people that you're scared of Shaq."
However, Beckham later rejected the offer, joking to TMZ: "I don't see him calling Kobe out... I think Kobe could score against him."
O'Neal later responded with another Twitter message, reading: "Official, today is David Beckham is scared of Shaq day, he should be scared, he can never score a goal on me."
O'Neal has already set up a string of challenges, including a swimming match against Olympic champion Michael Phelps, a tennis game with Serena Wlliams and a boxing round with Oscar De La Hoya.