Care requires surgery to the joint of his big left toe and will therefore be unavailable for selection when England team manager Martin Johnson names his 30-man squad on Monday.
"We are all hugely disappointed for Danny who has worked extremely hard and has been an important part of the squad throughout our Rugby World Cup preparations," said Johnson.
"He featured in both warm-up fixtures and was on course for selection so this injury is really disappointing for him and for us as a squad."
The setback is particularly unwelcome given the fact that fellow scrum-half Ben Youngs is currently recovering from a knee operation.
The Leicester star had keyhole surgery to trim cartilage in his left knee in June and, although he should be fit to start the final warm-up clash with Ireland on August 27, it is asking a lot of the 21-year-old to immediately produce his best form on his return.
Care had been expected to push Youngs hard for the No9 spot for the World Cup opener against Argentina on September 9 but that responsibility should now fall on the shoulders of Saracens' Richard Wigglesworth.
The former Sale man began the 19-9 defeat in Cardiff but came off after a blow to the head mid-way through the first half.
Wigglesworth now appears guaranteed to win a place in Johnson's squad for the global gathering in New Zealand, with full back Ben Foden likely to provide extra cover if Johnson opts not to take a third specialist No9.
Joe Simpson of Wasps placed could be placed on standby if he doesn't win a full recall having been dropped from the original 45-man training squad at the beginning of the month.