The Italian eatery was usurped by Eleven Madison Park in New York, the first American restaurant to take the title since 2004.
But Modenese chef Bottura still has plenty to celebrate, taking second place in the awards and remaining the 'best in Europe' according to the 900 judges.
The jury this year labelled Bottura “a poet, storyteller and artist as much as a cook”, for “weaving narravtives” through the dishes on offer.
Bottura's restaurant offers a creative twist on Italian favourites, with dishes including 'Yellow is bello' and 'Oops I dropped the lemon tart'.
Highlights on the menu include Five Ages of Parmiggiano Reggiano, where the region's famed cheese is served in different forms and textures, as well as classics like tagliatelle with hand-chopped ragu and risotto cooked with veal-jus.
As well as running the celebrated restaurant, Bottura works on projects to eliminate food waste and help tackle social issues through food. Last year, he opened up a restaurant in Rio de Janeiro, Brazil, to feed the city's homeless community using leftover food from Olympics caterers.
Four other Italian eateries were recognized in Wednesday's awards.
Enrico Crippa's Piazza Duomo in Alba won 15th place, earning praise for its salads using “varying seasonal ingredients” and an unusual, entirely pink dining room, complete with frescoes inspired by the local landscape.
Le Calandre in Rubano – run by Max Alajuno, the youngest ever chef to receive three Michelin stars – came in at 29th. Judges said the Paduan eatery's standout dish was its saffron and liquorice risotto and that the meals on offer were “relatively simple and, above all else, delicious”.
And at 43rd place was Reale in Abruzzo, praised as a “truly original restaurant”. Housed in a former monastery, chef Niko Romito – who previously led a campaign to improve the quality of Italian prison food – focuses on “complexity” in his food.
Organized by the British magazine ‘Restaurant Magazine' in collaboration with San Pellegrino and Acqua Panna, the awards showcase the 50 best restaurants which are selected by over 900 international jurors.