The third instalment of the DreamWorks animated series took in an estimated $60.35m across 4,258 theatres.
Ridley Scott's Prometheus finished in second place, bringing in $50m. However, the sci-fi project appeared at almost a thousand less cinemas than Madagascar 3.
Snow White and the Huntsman drops two to three from last weekend's number one, taking in a further $23m.
Will Smith's Men in Black III and The Avengers complete the top five, achieving $13.5m and $10.8m respectively.
The Avengers has now secured over $571m domestically, while Men in Black III has broken the $125m mark.
The Best Exotic Marigold Hotel and What to Expect When You're Expecting are level at six and seven, with Battleship and The Dictator each dropping five spots to eight and nine.
Wes Anderson's Moonrise Kingdom climbs three places to number ten.
The US box office top ten in full (studio estimates):
1. (-) Madagascar 3: Europe's Most Wanted - $60,350,000
2. (-) Prometheus - $50,000,000
3. (1) Snow White and the Huntsman - $23,021,000
4. (2) Men in Black III - $13,500,000
5. (3) The Avengers - $10,809,000
6. (6) The Best Exotic Marigold Hotel - $3,235,000
7. (7) What to Expect When You're Expecting - $2,710,000
8. (4) Battleship - $2,286,000
9. (5) The Dictator - $2,150,000
10. (13) Moonrise Kingdom - $1,579,000
Watch the trailer for Madagascar 3 below: