Beset by years of poor performance, the retailer's value has fallen below the threshold for inclusion.
The company's value is now pitched below the £4.2bn benchmark required to secure a place.
Once a stalwart of the British high street, changing shopping habits and the emergence of fast fashion have left M&S struggling to keep up.
The last three years have seen the company's value slide 45%.
Former Tory MP and ITV boss Archie Norman has been at the helm of the company since September 2017.
It's the second time the former environment secretary has steered a company out of the stock market's top tier - the first time was during his time working broadcasting.
But he remained adamant the end is not nigh for the business.
He said: "When I went to ITV we dropped out of the FTSE 100, the sky didn’t fall in - the business was the same business the day after."
The relegation is symbolic of Britain's ailing high streets. Retail analyst Helal Miah believes the food, clothing and sundries supplier's fall is "highly symbolic of the troubles on the UK high street and the challenges the UK retail sector faces as the internet plays an increasingly important role".
M&S may not be the last retail stalwart to find itself in the FTSE 250, with Sainsbury’s, Morrisons and B&Q owner Kingfisher all set to be contenders for relegation in the next reshuffle.