Coach Bert van Marwijk said the Hamburg forward was now fit but that a long-term absence had ended his chances.
Van Nistelrooy last featured for the Netherlands at the 2008 European Championship. Though the striker wanted to play in South Africa, Van Marwijk said he wasn't at his best after injuring his knee while playing for Real Madrid.
"I have told him that we have intensely followed him and we have admiration for his desire and efforts to go to the World Cup," Van Marwijk said. "As far as I know, Ruud is fit. But there is insufficient time for him to get to his normal levels after his lengthy knee injury."
There were no major surprises in the 30-man squad, which included Liverpool duo Ryan Babel and Dirk Kuyt, along with John Heitinga, Nigel de Jong and Robin van Persie.
The Dutch squad is:
Goalkeepers: Maarten Stekelenburg (Ajax), Sander Boschker (FC Twente), Michel Vorm (Utrecht).
Defenders: Andre Ooijer (PSV Eindhoven), Vurnon Anita (Ajax), Khalid Boulahrouz ( Stuttgart), Edson Braafheid (Celtic), Giovanni van Bronckhorst (Feyenoord), John Heitinga (Everton), Joris Mathijsen (Hamburg), Ron Vlaar (Feyenoord), Gregory van der Wiel (Ajax).
Midfielders: Wesley Sneijder (Inter Milan), Otman Bakkal (PSV Eindhoven), Mark van Bommel (Bayern Munich), Wout Brama (FC Twente), Orlando Engelaar (PSV Eindhoven), Ibrahim Afellay (PSV Eindhoven), Nigel de Jong (Manchester City), Stijn Schaars (AZ Alkmaar), David Mendes da Silva (AZ Alkmaar), Rafael van der Vaart (Real Madrid), Demy de Zeeuw (Ajax).
Strikers: Arjen Robben (Bayern Munich), Ryan Babel (Liverpool), Eljero Elia (Hamburger SV), Dirk Kuyt (Liverpool), Jeremain Lens (AZ Alkmaar), Robin van Persie (Arsenal), Klaas Jan Huntelaar (AC Milan).
Meanwhile, England's Group C opponents Slovenia have kept faith with the core of players who ensured they were one of the World Cup's shock qualifiers.
West Brom's Robert Koren is the only British-based player in coach Matjaz Kek's provisional 30-man finals panel.
Newcastle midfielder Haris Vuckic (17), and ex-captain Klemen Lavric miss out despite speculation they would feature.
Goalkeepers: Samir Handanovic (Udinese), Jasmin Handanovic (Mantova), Aleksander Seliga (Sparta Rotterdam), Jan Koprivec (Maribor).
Defenders: Bojan Jokic (Chievo Verona), Marko Suler (Ghent), Bostjan Cesar (Grenoble), Branko Ilic (Lokomotiv Moscow), Matej Mavric (Koblenz), Dejan Kelhar (Cercle Brugge), Elvedin Dzinic (Maribor), Miso Brecko (Cologne), Aleksandar Rajcevic (Koper), Suad Filekovic (Maribor).
Midfielders: Andraz Kirm (Wisla Krakow), Andrej Komac (Maccabi Tel Aviv), Rene Krhin (Inter Milan), Mirnes Sisic (Giannina), Darjan Matic (Rapid Bucharest), Dare Vrsic (Koper), Dalibor Stevanovic (Vitesse Arnhem), Robert Koren (West Bromwich Albion), Aleksander Radosavljevic (Larissa), Valter Birsa (Auxerre).
Forwards: Milivoje Novakovic (Cologne), Zlatko Dedic (VfL Bochum), Zlatan Ljubijankic (Ghent), Nejc Pecnik (Nacional), Miran Burgic (AIK Solna), Tim Matavz (Groningen).