The venerable footwear was uncovered by maintenance staff of the St John's College while they were removing panels to install electrical cables a common room for senior academics.
The building was originally the residence of the Master of the College.
It was built between 1598 and 1602, but it is thought that the shoe was put behind the panels during changes to its interior, between the end of the 17th Century, and mid-way through the 18th, the university said in a statement.
The discovery of the left shoe of a man, measuring over nine and a half inches (about a size 6 by modern standards) has been referred to the Cambridge Archaeological Unit, who have taken photographs and are consulting a specialist in the hope of establishing a precise date.
Richard Newman, from the Archaeological Unit, described it as a "classic example" of what researchers call "apotropaic" magic, popular magic designed to bring luck and turn away evil influences.
"Given its location, it is very likely that it was there to play a protective role for the Master of the College. It may even have been one of his old shoes," he said.
Popular magic of this type was fairly widespread in England from the 16th to mid-19th Centuries, especially in East Anglia, according to the statement.
Although shoes were the most common item of choice for keeping spirits at bay, they were far from the only object embedded in walls for this purpose.
They were hidden in walls, roofs and beneath floors to prevent malevolent forces from bringing bad luck to the building, or its occupants.
Why shoes were such a popular choice is not properly understood. One theory is that they were believed to be an effective "spirit trap".
Perhaps more plausibly, some historians argue that, at least in the days before mass-production of leather shoes, they took on something of the shape of the wearer's foot.
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