On August 3, the Protector Trials pack will have players defend Little Sisters against swarms of enemies in a variety of challenge rooms.
The content will contain six maps based on locations from the main game, alongside three difficulty levels, seven Achievements / Trophies, and new concept art and videos to unlock.
2K Games president Christoph Hartmann also said that it will be the first of many downloadable packs, adding: "The team at 2K Marin has been working hard ever since the launch of BioShock 2 to develop ways for gamers to extend their time in the single-player world of Rapture.
"I'm proud to announce the first of these experiences, the Protector Trials, which takes players back to some of their favourite places from the original game and features some of the biggest challenges that fans of this series have ever experienced.
"This is just the beginning. More single-player content is on the way that will deliver more of the intense action and story-driven gameplay that fans have been craving."
The pack will be available on Xbox 360 and PC for 400 Microsoft Points (£3.40) and PlayStation 3 for $4.99 (£3.50).
> Click here to read our review of BioShock 2