The Dutch football federation confirmed the news on Monday, stating that Timber "has not recovered sufficiently from a groin injury to take part in the World Cup in a medically responsible manner" and that the decision was made in consultation with the medical staff.
Timber's withdrawal was confirmed in an emotional Instagram post by the 24-year-old himself.
"Heartbroken to miss the World Cup. Representing my country on the biggest stage is something I've always dreamed of, and it's difficult to accept that injury has taken that opportunity away from me."
He added: "While this isn't the path I had hoped for, I trust that God has a plan greater than the one I can see right now. Wishing the boys all the best. Make the nation proud!"
Netherlands head coach Ronald Koeman had included Timber in the squad despite the defender playing only limited football since March, when the groin problem first forced him out of Premier League action. Timber returned to feature as a substitute in Arsenal's Champions League final defeat to Paris Saint-Germain — approximately 60 minutes of football including extra time — but the recovery has not progressed as hoped in the weeks since.
Sunderland defender Lutsharel Geertruida has been named as Timber's replacement. The 25-year-old had featured in the Netherlands' squad as recently as the June 3 warm-up match against Algeria and will slot straight into the defensive setup.
Koeman addressed reporters after the decision was confirmed.
"With Jurriën in the squad, we have eight defenders; without him, there are seven, and that's not enough. So you have to make a decision. We had Geertruida with the squad right up until the match against Algeria, so it wouldn't be too much of a struggle to bring him in. We didn't wait any longer because his situation wasn't expected to improve any time soon."
The Netherlands open their World Cup campaign on June 14.
Timber will watch it from home.