The pair of ebonised oak chairs, which date from 1903, are going under the hammer in Edinburgh next month.
They were made for Miss Cranston's Willow Tea Rooms in Glasgow but are being sold by a private seller.
Kate Cranston was one of Mackintosh's most consistent patrons and between 1897 and 1917 he designed or restyled rooms in all four of her Glasgow tea rooms.
The chairs, valued at £10-15,000 will be part of auctioneers Lyon & Turnbull's Scottish Design sale being held on August 13.
John Mackie, director and head of decorative arts, said: "The chairs were used by Mackintosh to divide the space in the tea rooms and form an intimate environment within a much larger space.
"The chairs' design is informed by vernacular 'ladder back' chairs from the past; however Mackintosh has taken this traditional chair form and made it his own.
"The simplicity of line and striking black colour juxtaposed with the traditional rushed seat creates a powerful visual impact and makes this chair one of Mackintosh's most recognisable designs."
The private London-based seller said: "I feel that I have been extraordinarily lucky as I was given the chairs by a friend when I was 21 years old, in the early 1960's.
"Since then I have used them every day, but after some careful thought and discussions with my wife, we have decided to sell the chairs as we would like others to enjoy them as much as we have."
The Willow Tea Rooms in the city's Sauchiehall Street remain a major attraction.