The brand's campaign is headed by England rugby star Lewis Moody, Welsh player Lee Byrne and Scottish international Sean Lamont.
The three players will produce exclusive video diaries and appear on point-of-sale branding in bars and shops during the tournament.
The deal marks the first time the 100-year-old tournament has signed an official beer partner.
Guinness already has a long-standing association with the sport. From 2005 until last year .
Guinness also has existing sponsorship deals with the respective rugby unions of England, Wales, Scotland and Ireland.
No financial details of the sponsorship deal, which was overseen by John Feeham, chief executive of RBS Six Nations Rugby, have been disclosed.
The deal has no impact on RBS's headline sponsorship of the event.
The tournament kicks off on 4 February when England play Wales.
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