The Pond-Moonlight - taken in New York in 1904 - was sold for $2.9m (£1.6m), more than doubling the previous record.
The 41x48cm photo shows a pond in a wooded area with light coming through the trees and reflected in the water.
The only other two copies in existence are in museum collections. The buyer's identity has yet to be disclosed.
But no doubt he or she feels that owning a piece of photographic history is more than worth the record-breaking price-tag, the BBC's Elettra Neysmith says.
The photograph was put up for sale by the Metropolitan Museum of Art, which has another copy in its collection.
Stephen Perloff, the editor of the Photograph Collector, a newsletter about the photography art market, said before the Steichen auction that it would be a "moment of history", the Associated Press news agency reports.
Steichen began experimenting with colour photography in 1904.
He was one of the first Americans to use the autochrome - a revolutionary method of producing colour photographs.
The previous record for highest price for a photograph at auction was set last November by Richard Prince's Untitled (Cowboy). It sold for $1,248,000 (£719,000).
The Pond-Moonlight was auctioned at Sotheby's in New York.