The Hamburg captain was hoping to feature at his third World Cup as the Netherlands aim to improve on their final defeat to Spain at the 2010 event in South Africa.
But the 31-year-old has not been able to shrug off a calf issue, with Louis van Gaal's decision to leave him out of his final 23-man squad being announced on Wednesday.
A KNVB statement read: "Rafael van der Vaart is missing the World Cup in Brazil.
"The Dutch national team player has a (strained) calf muscle in his right leg.
"National coach Louis van Gaal has decided not to include him in the final selection.
"This leaves Van der Vaart provisionally on 109 international games. He was the most experienced player in the current pre-selection."
Van der Vaart - formerly of Real Madrid and Tottenham - helped Hamburg escape relegation by the narrowest of margins in the recently completed Bundesliga season.
A 1-1 aggregate draw with second-tier Greuther Furth in their end-of-season play-off saw Hamburg retain their top-flight status by virtue of the away goals rule.
