Discolouration and dirt had concealed the double-sided painting by Italian Renaissance artist Pietro di Niccolo da Orvieto, displayed at the Fitzwilliam Museum in Cambridge.
Seven painting conservators from the Hamilton Kerr Institute brought the 15th century portrait back to life.
According to the museum, the painting is "testament to the religious devotion of the Italian Renaissance".
The portrait had been displayed for years without anyone realising it was double-sided. The concealed side of the picture is believed to depict Christ's crucifixion wound.
The painting now sits in a glass display at the museum where both sides can be seen, as part of the Madonnas and Miracles exhibition.
Madonnas and Miracles: The Holy Home In Renaissance Italy is on at the Fitzwilliam Museum in Cambridge until 4th June.