The Academy Awards host takes the role of a bartender in a clip from The World is Not Enough, Brosnan's third movie as the secret agent.
MacFarlane opens the clip by promising that this year's Academy Awards will celebrate "all your favourite films - like the James Bond movies".
His remark references reports that Oscars chiefs want to reunite all six actors who have played 007, in order to recognise the recent 50th anniversary of the series on screen.
MacFarlane's bartender is later seen chiding Bond when he orders his usual "vodka martini, shaken, not stirred", saying: "No-one has ever stirred a martini. That's not how you make a martini."
He then asks Bond whether it's his first martini, assuring the spy: "Yeah, you're gonna have fun."
MacFarlane previously appeared in an Oscars promo referencing Alfred Hitchcock horror Psycho, in which the Family Guy creator is seen banging on the wall of his hotel room and shouting at Janet Leigh to "keep it down".
The 2013 Academy Awards will be held on Sunday, February 24 at Hollywood's Dolby Theater.
Oscars hosts through the years - Photo gallery: