On 30 January 1649, the monarch was beheaded in front of a crowd of onlookers outside the Banqueting House in Whitehall, London.
The king wore a pale blue, silk vest for his beheading, which is due to be displayed later this year as part of an exhibition called â€œExecutionsâ€.
Following King Charles Iâ€™s execution, which was ordered after he was found guilty of treason, the items of clothing he was wearing were given out to the crowds.
Meriel Jeater, curator at the Museum of London, said it is â€œexcitingâ€ to be able to feature â€œthis incredibly rare vestâ€ in the exhibition, which will explore executions that took place in London from 1196 to 1868.
â€œIt is key in telling the story of one of the most infamous executions that occurred in the capital,â€ Jeater said.
â€œThe exhibition covers nearly 700 years, a time when public executions were more frequent in London than in any other town, attracting huge crowds several times a year at locations across the capital.â€
When the Museum of London received the vest in 1925, it came with a note validating its authenticity as the garment worn by the monarch for his execution.
The vest is made from knitted, high-quality silk, and features stains on the front that the museum says could be bodily fluids.
The vest is not the only item of clothing worn by King Charles I for his beheading that is to go on display at the Museum of London.
Other items of the king also featured in the exhibition include a pair of gloves, fragments from a cloak, a sash and a handkerchief.
The exhibition is due to open in October, with tickets going on sale on the Museum of London website from Saturday 1 February.
Earlier this month, it was reported that several items from the wardrobe of Queen Victoria were going to be auctioned, having been safeguarded in a wardrobe for a century.
Following the death of Queen Victoria in 1901, royal photographer Alexander Lamont Henderson were given several of her sartorial possessions, including a pair of bloomers, a bodice, a skirt and a pair of leather boots.
Having been passed down through generations, the photographerâ€™s great-great grandson decided to sell the collection, which was predicted to be sold for more than Â£15,000.