In the 24th century the exploitation of the Earth's ecosystem has created an environment so toxic that mass extinctions have wiped out nearly half of its animal population. Most of the remaining fauna, save humans, have suffered horrible mutation. Aided by the benevolent and technologically superior alien race, the Yma, humanity begins to repair their ravaged world while simultaneously learning more about the universe around them. When an unmanned Yma probe discovers evidence of alien life on another planet, the titular "expedition" is sent to investigate.
Barlowe writes as a sort of 24th century Audubon, presenting his findings in a collection of paintings, sketches, field notes, and diary entries from his explorations of Darwin IV. He details a bewildering variety of alien lifeforms such as Gyrosprinters, Arrowtongues, Grovebacks, Daggerwrists, Skewers, Emperor Sea Striders, and Eosapians. Unlike the aliens presented in much of popular science fiction, which often seem to be variations of terrestrial lifeforms, Barlowe's creatures are truly alien: none of them possess eyes or true jaws; their body structures are often unlike any found on Earth; and they have unique modes of locomotion, sensing, and eating. Very late in the expedition, the explorer encounters lifeforms which use tools (the Eosapiens), giving a very strong indication they are intelligent.
A conservationist theme is present throughout the book. The expedition is designed to have as minimal an impact as possible on Darwin IV's environment. When two of the expedition's members suffer a fatal accident, Yma technology is used to remove all traces of the accident from Darwin IV's environment. At the conclusion of the expedition, Darwin IV is left in the same pristine state it was in prior to the expedition, with the exception of a metal obelisk placed in a remote area by the expedition.
The Discovery Channel produced a television special adapted from Barlowe's Expedition, entitled Alien Planet, which first aired on May 14, 2005. This program was faithful to the book in its presentation of the lifeforms found on Darwin IV. However, instead of being presented as the artist's own experiences, the program is presented as the findings of two autonomous robotic probes.