Chief executive Stuart Rose was presented with the award by HRH The Prince of Wales.
Prince Charles said: 'M&S has had a hugely successful year on the high street and has not only reinvented itself, but in doing so has never sacrificed its commitment to responsible business practice, indeed it has continued to lead the way.'
This year, Marks & Spencer guaranteed British dairy farmers a fixed price for their milk and became the first major UK retailer to introduce fair trade cotton clothing into its stores. The retailer also picked up the award in 2004.
Boots was a runner-up for the top prize, while Waitrose won the award for the most responsible supply chain and Tesco was highly commended for its Computers for Schools programme.
John Lewis Partnership chairman Sir Stuart Hampson was appointed as a National Ambassador for the year, for his work championing community regeneration.