El Celler de Can Roca toppled Denmark's Noma from its position to claim the title after spending two years as runner-up on the World's 50 Best Restaurants list - billed as the Oscars of the restaurant world.
The Roca brothers' avant-garde eatery in Girona, north east Spain, now heads a top-10 lineup dominated by Spanish cuisine.
Dinner By Heston Blumenthal - the chef behind The Fat Duck - was named Britain's top offering.
Positions two and three on this year's list were taken by chef Rene Redzepi's Noma, in Copenhagen, which has famously served moss, lichen and bone marrow, and Massimo Bottura's comparatively low-key Osteria Francescana in Modena, northern Italy.
El Celler de Can Roca, run by the three Roca siblings, is well-known for its free-style cooking and has long been hailed for its combination of Catalan dishes and cutting edge techniques.
With Joan Roca heading up the kitchen and his brothers Jordi and Josep as head pastry chef and head sommelier respectively, it has built up a reputation as one of Spain's most exciting places to eat.
Other Spanish restaurants to make it onto the list this year include San Sebastian's Mugaritz at number four, and Arzak, which remained at number eight.
The Alicante restaurant Quique Dacosta, famed for its eco-cuisine, came in at number 26 while Asador Etxebarri, in the tiny leafy village of Axpe, set between San Sebastian and Bilbao, took 44th position.
El Bulli, the restaurant in Catalonia which achieved the top ranking five times, does not appear after chef Ferran Adria announced a two-year closure.
France and America were the two countries with the most restaurants on the list, both notching up six in the top 50.
Culinary maestro Alain Ducasse, who became the first chef to earn three Michelin stars for three different restaurants, was honoured with this year's Lifetime Achievement Award.
The list was drawn from the votes of more than 900 leading figures in the industry - including food critics, chefs, restaurateurs and highly regarded "gastronomes".