The shuttle was set to touch down early Thursday at 5:56 am (0956 GMT), 42 years after US astronaut Neil Armstrong became the first person to step foot on the Moon as part of the Apollo 11 mission.
Atlantis's landing will end an era of US dominance in human space exploration, leaving Russia as the sole taxi to the International Space Station until a replacement US capsule can be built by private industry.
The crew of four US astronauts aboard Atlantis on the mission known as STS-135 savored their final hours in orbit after a successful trip to restock the ISS for a year with several tons of supplies and food.
Final inspections of the shuttle's heat shield, which protects the spacecraft during its fiery transition into Earth's atmosphere, were completed Wednesday and NASA said the spacecraft looked to be in good shape for landing.
The weather forecast was also "very favorable" at Kennedy Space Center, NASA said.
The crew was set to awaken at 9:29 pm (0129 GMT) and the shuttle was scheduled to begin its deorbit burn at 4:49 am (0849 GMT) to re-enter the Earth's atmosphere.
If conditions do not allow for the first attempt, a second opportunity for deorbit would begin at 6:25 am (1025 GMT) with the landing at 7:32 am (1132 GMT).
"Forty-two years ago today, Neil Armstrong walked on the moon and I consider myself fortunate that I was there to actually remember the event," Atlantis commander Chris Ferguson said to mission control, recalling the images of July 20, 1969.
"It is kind of interesting to be here on the final night of the shuttle mission. We don't quite know what to think. We are just trying to take it all in."
Ferguson then read a quote by Apollo-era flight director Gene Kranz, best known for leading mission control's successful effort to save the Apollo 13 astronauts after an oxygen tank exploded on a trip to the Moon.
"I pray that our nation will someday find the courage to accept the risk and challenges to finish the work that we started," the commander said, calling the quote by Kranz, whose hero role was showcased in a 1995 movie starring Tom Hanks as an astronaut and Ed Harris as Kranz, "very appropriate."
Over the course of the program, five NASA space shuttles -- Atlantis, Challenger, Columbia, Discovery and Endeavour -- have comprised a fleet designed as the world's first reusable space vehicles.
The first shuttle flight to space lifted off April 12, 1981.
Columbia and Challenger were destroyed in accidents that killed their crews, leaving only three in the space-flying fleet and Enterprise, a prototype that never flew in space. The quartet will become museum pieces in the coming months.
Critics have assailed the US space agency for lacking a focus with the space shuttle gone and no next-generation human spaceflight program to immediately replace it.
The astronaut corps now numbers 60, compared to the 128 employed in 2000, and thousands of people are being laid off from Kennedy Space Center. But NASA chiefs say future missions to deep space should revive hope in the US program.
"We have just not done a good job of telling our story. NASA is very busy," the space agency's administrator Charles Bolden told CNN.
"The president said to us, 2025 for an asteroid and 2030 to Mars. We have a lot of work to do ahead."
Mission specialist Rex Walheim said, as the crew sat for a series of TV interviews, that he was optimistic about the US space program, but acknowledged "we're in a kind of a transition period, which is a little bit uncomfortable."
NASA aims to turn over low-orbit space travel and space station servicing to commercial ventures, with a commercial launcher and capsule built by a private corporation in partnership with NASA ready to tote crew members after 2015.
Until the private sector fills the void, the world's astronauts will rely on Russian Soyuz rockets for rides to the ISS.
NASA flight director Tony Ceccacci said his team was just trying to keep emotions at bay and focus on getting the shuttle home safely.
"We have a motto in the mission control center that flight controllers don't cry, so we are going to make sure that we keep to that."