During the planned 11-day mission, Wilcutt and his crew mates spent a week inside the ISS unloading supplies from both a double SPACEHAB cargo module in the rear of Atlantis's cargo bay and from a Russian Progress M-1 resupply craft docked to the aft end of the Zvezda Service Module. Zvezda, which linked up to the ISS on 26 July, served as the early living quarters for the station and is the cornerstone of the Russian contribution to the ISS.

Mission STS-106 was added to the manifest after delays in launching Zvezda. The STS-101 flight was originally planned to carry cargo to the ISS and have three crew perform an EVA to connect Zvezda to the ISS, but the delays caused the mission objectives of STS-101 to be split into 2A.2a (STS-101) and 2A.2b (STS-106). The three spacewalk crewmembers Lu, Williams, and Malenchenko followed their EVA onto STS-106.
The goal of the flight was to prepare Zvezda for the arrival of the first residents, or Expedition, crew later in the fall of 2000 and the start of a permanent human presence on the new outpost. That crew, made up of Expedition Commander Bill Shepherd, Soyuz Commander Yuri Gidzenko and Flight Engineer Sergei Krikalev, launched on 31 October 2000 in a Soyuz capsule from the Baikonur Cosmodrome in Kazakhstan for a four-month "shakedown" mission aboard the ISS.

On flight day three, Dr. Ed Lu and Yuri Malenchenko (Col., Russian Air Force), who were both making their second flights into space, conducted a 6-hour and 14 minute space walk. The spacewalk's objective focused on routing and connecting nine power, data and communications cables between the Zvezda module and the other Russian-built module, Zarya, as well as installing the six-foot-long magnetometer. The magnetometer would serve as a three-dimensional compass designed to minimize Zvezda propellant usage by relaying information to the module's computers regarding its orientation relative to the Earth.

Lu and Malenchenko used tethers and handrails along the ISS to make their way to a point more than 100 feet above the cargo bay, the farthest any tethered spacewalker has ventured outside the shuttle. They completed this with the assistance of their crewmates Burbank and Mastracchio who deftly maneuvered them around with the robotic arm. This spacewalk celebrates the sixth spacewalk in support of the station assembly and the 50th spacewalk in Space Shuttle history. Also this was the second joint U.S.-Russian space walk outside a Space Shuttle, following on the work conducted by Astronaut Scott Parazynski and Cosmonaut Vladimir Titov outside Atlantis while docked to the Mir Space Station during the STS-86 mission in October 1997. Lu, designated EV 1, wore the space suit marked by red stripes, while Malenchenko, EV 2, wore the pure white suit. This was Lu's first space walk, while Malenchenko had conducted a pair of space walks totaling 12 hours during his four-month stay aboard Mir in 1994. Dan Burbank (Lt. Cmdr, USCG), who was a spaceflight rookie, served as the space walk choreographer.

Mission Specialist Rick Mastracchio, also a spaceflight novice, was the prime robot arm operator for the mission, using the Canadian-built arm to move Lu and Malenchenko around the ISS as they conducted their assembly work. Mastracchio is backed up on arm operations by Pilot Scott Altman (Cmdr., USN), making his second flight into space.

The final member of the crew was Russian Cosmonaut Dr. Boris Morukov, making his first flight into space. Morukov was responsible for unloading supplies from the Progress vehicle during the docked phase of the flight.