The illustration of the bear playing Poohsticks with Piglet and Christopher Robin was first published in 1928 and has been in a private collection for more than 40 years. But the ink drawing by EH Shepard will now be sold by Sotheby's next month, reported BBC online.
The illustration featured in AA Milne's book The House At Pooh Corner. It accompanied the closing scene of Chapter 6, "in which Pooh invents a new game and Eeyore joins in."
The drawing will feature in Sotheby's sale of English literature, history, children's books and illustrations on December 9 and is estimated to fetch between 100,000 pounds and 150,000 pounds.
The auction house said, "The illustration can be counted among the most familiar and most loved cultural references in English literature."
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The original wooden crossing on which the illustration is based - known as Posingford bridge, at Hartfield farm, East Sussex - had fallen into disrepair by the 1970s.
It was completely rebuilt in 1999.