The 23-year-old met up with the rest of the squad at their team base in Watford on Sunday night but after analysis from the England medical staff, he was deemed unfit to play.
"Kyle Walker has withdrawn from the England squad due to a groin injury," a Tottenham statement read.
"The full-back has missed our last four matches with the problem and will not be fit for England's friendly against Denmark on Wednesday."
Walker's absence means Glen Johnson is likely to win his 50th cap against the Danes at Wembley.
Walker, Andros Townsend, Steven Caulker, Adam Lallana and John Ruddy all missed England training on Monday.