The 3D movie, featuring the voices of Jesse Eisenberg and Anne Hathaway, edges past Tyler Perry's latest movie Madea's Big Happy Family by just over $1 million to secure the top spot.
Water for Elephants debuts at number three with £16m, making it the most successful non-Twilight opening for Robert Pattinson. The British star's Remember Me bowed to a disappointing $8m last March.
Russell Brand's Hop and slasher sequel Scream 4 complete the top five, while African Cats - the latest movie in Disneynature 'Earth Day' (April 22) annual releases - registers at number six with $6m ticket sales.
Elsewhere, action-thriller Hanna is the biggest chart faller as it slips four spots to number eight.
The US box office in full (Hanna figures are studio estimates):
1. (1) Rio - $26,323,321
2. (-) Tyler Perry's Madea's Big Happy Family - $25,068,677
3. (-) Water for Elephants - $16,842,353
4. (3) Hop - $12,185,905
5. (2) Scream 4 - $7,030,747
6. (-) African Cats - $6,003,200
7. (5) Soul Surfer - $5,436,868
8. (4) Hanna - $5,277,000
9. (7) Insidious - $5,207,633
10. (8) Source Code - $5,091,347
Watch the first two minutes of Rio below: