In the two-minute clip, which will premiere on ITV on Friday evening, the Pulp Fiction and Marvel star, 74, can be seen impersonating the chairman of the baking company, Jonathan Warburton.
Jackson said it was a “pleasure” to meet the man at the helm of Britain’s biggest family bakery.
“And what an honour to follow in the footsteps of ‘Bolton alumni’ George Clooney and Robert De Niro,” he said.
“We had a lot of fun on set so I hope people enjoy the finished product – the ad and toastie loaf.”
Mr Warburton said of the advert: “Samuel L Jackson was a pleasure to work with and is welcome back at our bakeries any day.
“Not many people can be that commanding and so charming at the same time, and I love his hilarious take on why our toastie loaf is the real deal.
“Quality will always be at the core of our fifth-generation family run business, and our new ad highlights this commitment to freshness and quality in all that we bake.
“Inviting the big-screen hero of Samuel L Jackson into the business was a uniquely memorable experience, and we hope to bring some light-hearted humour to viewers at home – while reminding the nation that our toastie truly offers our customers the best of the best.”
The advert opens in Warburtons HQ in Bolton where Jackson is seen taking over the office of chairman Mr Warburton.
Dressed in a blue shirt and orange tie, Jackson says: “Hi, I’m Jonathan Warburton, chairman of the UK’s largest family bakery.”
Looking to camera, Jackson then shares a social post in which a person called Steve has questioned the similarity of Warburtons’ much-loved toastie loaf with other shelf contenders.
This prompts anger from Jackson, who says: “Hell hath no fury like a baker scorned, Steve.”
A montage then follows which shows the effort that goes into making the perfect toastie loaf before Jackson appears on the doorstep of Steve’s parents house at breakfast time, where he swaps out their toast for the Warburtons toastie loaf.
Also in the advert, Jackson is seen testing for softness, butter-ability and bounce-back-ability.
The clip ends with the Hollywood star knocking down the door of the chairman’s office where he finds the real Mr Warburton, who says: “I couldn’t have said it better myself.”
The bread-baking business is run by the fifth-generation of Warburtons, having been set up in 1876 as a grocery shop in Bolton by Thomas and Ellen Warburton.
The new advert will go live on social media on Thursday and will premiere on ITV 1 on Friday evening.