The 33-year-old has been out since breaking a bone in his right shoulder against Australia in November.
However, he has been included in the South Africa A four-day squad for their upcoming tour of England.
Paceman Vernon Philander also misses out on the Champions Trophy in England and Wales, which starts on 1 June.
The Sussex all-rounder picked up a groin injury in their County Championship defeat by Kent over the weekend.
Left-arm spinner Keshav Maharaj has earned his first one-day international call-up while, with Steyn and Philander out, pace bowler Morne Morkel makes a return to the limited-overs format after nearly 10 months.
AB de Villiers is fit to captain the side, despite injuries disrupting his participation at the Indian Premier League.
Selection convener Linda Zondi told told Cricket South Africa's website: "We have been working with this group of players for the last two seasons where our key emphasis has been consistency in selection.
"That has shown in how this group has performed over the last while, which has resulted in winning three consecutive ODI series."
The Proteas will play three ODIs against England, starting with the first match at Headingley on 24 May, before their first Champions Trophy group match against Sri Lanka at The Oval on 3 June.
After the Champions Trophy, South Africa remain in England to play four Tests, starting on 6 July - and Steyn could be selected for that squad if he proves his fitness.
South Africa squad for three-match ODI series against England and ICC Champions Trophy: Hashim Amla (Cape Cobras), Farhaan Behardien (Titans), Quinton de Kock (Titans, wk), AB de Villiers (Titans, capt & wk), Faf du Plessis (Titans), JP Duminy (Cape Cobras), Keshav Maharaj (Dolphins), David Miller (Knights), Morne Morkel (Titans), Chris Morris (Titans), Wayne Parnell (Cape Cobras), Andile Phehlukwayo (Dolphins), Dwaine Pretorius (Highveld Lions), Kagiso Rabada (Highveld Lions), Imran Tahir (Dolphins).
Pace bowler Dale Steyn has been left out of South Africa's ICC Champions Trophy squad as he continues his recovery from a shoulder injury.