Ralph, who lived at Marwell Zoo, passed away aged 19, the wildlife park confirmed on Twitter.
In a post staff said he was diagnosed with arthritis eight months ago, which had been managed by medicine until recently.
They added that they had taken ‘the difficult decision’ to euthanize him to stop ‘any unnecessary distress or pain’.
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Ralph made international news when he started to wear a wetsuit as protection against the elements.
Park staff found he moulted at a quicker rate than other penguins, leaving him with sensitive bald patches.
James Ellis, birds team leader at Marwell Wildlife, said: ‘It is very sad that we have had to say goodbye to Ralph, the oldest penguin in our colony.
‘Ralph was simply unique and never far from the spotlight with his mischievous antics, which will be sorely missed by everyone who knew and looked after him.
‘Ralph had a long and happy life and everyone has wonderful memories of him.’
Ralph and his partner Coral raised several chicks at Marwell, including Calippo who remains in the colony.
The wildlife park has asked people to have memories of Ralph to share them on its Twitter page.