Holley's side will launch their defence of the title they won in such dramatic fashion against Leinster at the RDS, Dublin at the end of May with a trip to face Ulster under the Ravenhill floodlights on Friday, 3 September.
Tommy Bowe grabbed a bonus point try in a 38-27 triumph in Belfast in April in what was one of four Ospreys wins in five games on Irish soil last season. That away success was vital in earning them a home Play-Off clash with Glasgow Warriors and they then went on to beat Leinster 17-12 on their own patch in the Grand Final.
"We have been champions three times - and that is really satisfying - but you cannot rest on your laurels," said Holley.
"Since the inception of Celtic League rugby in 2001, no champions have been able to retain the title. So we want to be proud champions and successfully defend our title in what appears to be the most difficult season to date to do that.
"That is a great challenge - but we know it is achievable and we want to sustain success at the Ospreys so we have to front up.
"Is it tougher to defend a title than win it? I suppose that as champions everyone wants to knock our block off and, while that is a compliment, it is also a good position to be in. We have got the yellow jersey and the others have to come and take it from us because this is a place where we want to stay."
The arrival of the two Italian teams means a 22-match fixture list ahead of the hugely successful Play-Offs and then the Grand Final in May with Holley accepting his side face a tough task in trying to claim that slice of Celtic Rugby history.
The Ospreys host Italian champions Bennetton Treviso in their first home fixture in the second round and Holley believes the introduction of the two new teams will further enhance the quality and interest in a tournament going from strength to strength.
"Last year showed what a competitive league the Magners is - and it is going to get even harder. The two Italian teams are slightly unknown quantities at this level but you saw the emergence of Glasgow as a real dominant force and Cardiff Blues are probably a bit disappointed they didn't figure as they should have last year," warned Holley.
"Those clubs will come strong again and Ulster have recruited really well. Then there are the usual suspects - and we like to think we are amongst them - with Munster and Leinster never far away as previous winners.
"There have also been positive signs elsewhere. Connacht and the Dragons had great seasons last year, and I am sure the Scarlets are determined to bounce back.
"It really is a wide open league and the crucial fixtures are probably those in the international windows - who you play then, what form you hit and how your squad functions."
Magners League Champions
2002 - Leinster
2003 - Munster
2004 - Scarlets
2005 - Ospreys
2006 - Ulster
2007 - Ospreys
2008 - Leinster
2009 - Munster
2010 - Ospreys
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