Launch from Cape Canaveral (KSC); night landing on Cape Canaveral (KSC). The launch was delayed three times due to high winds. Third Space Station Assembly mission ISS-03-2A.2A.

Following a two day solo flight the Atlantis docked with the ISS on May 20, 2000. The primary mission objectives for STS-101 was to deliver supplies to the International Space Station, with the supplies hauled up using a Spacehab double module and Spacehab Integrated Cargo Carrier pallet, perform a spacewalk and then reboost the station from 230 to 250 statute miles (370 to 402 km).

Detailed objectives included ISS ingress/safety to take air samples, monitor carbon dioxide, deploy portable, personal fans, measure air flow, rework/modify ISS ducting, replace air filters, and replace Zarya fire extinguishers and smoke detectors. Critical replacements, repairs and spares were also done to replace four suspect batteries on Zarya, replace failed or suspect electronics for Zarya's batteries, replace Radio Telemetry System memory unit, replace port early communications antenna, replace Radio Frequency Power Distribution Box and clear Space Vision System target.

James Voss and Jeffrey Williams performed the only EVA on May 21, 2000 (6h 44m). They installed the final parts of the Russian built crane, replaced a faulty antenna and installed some handrails and a camera cable.

The crew had to do several repairing work (replacement of batteries) in the ISS.

This mission was almost similar to the Columbia disaster. A damaged tile seam caused a breach which allowed superheated gas to enter the left wing during reentry. The gas did not penetrate deeply and the damage was repaired before the next flight. If it had penetrated deeply the Shuttle could have been destroyed during reentry.