1. Beat the game once on any difficulty. This will unlock Griff.
2. Beat the game again on any difficulty with a Silver Medal (25% accuracy). This will unlock Shadow Wolf.
3. Beat the game again on any difficulty with a Gold Medal (50% accuracy). This will unlock Pig Josh.
4. Beat the game again on any difficulty with a Platinum Medal (75% accuracy). This will unlock Annie Stoakes.
5. Beat the game once on Expert difficulty. This will unlock Buffalo Soldier.
6. Beat the game again on Expert difficulty with a Silver Medal (25% accuracy). This will unlock Ugly Chris.
7. Beat the game again on Expert difficulty with a Gold Medal (50% accuracy). This will unlock Dirty Steve.
8. Beat the game again on Expert difficulty with a Platinum Medal (75% accuracy). This will unlock Jack Swift.
9. Collect all 25 Hidden Horseshoes. This will unlock Red Harlowe, the main character.
Note: You do not need to beat the game on a higher difficulty to unlock characters from a lower difficulty. For example, you can beat the game on Normal difficulty to unlock Griff, then beat it on Hard difficulty to unlock Shadow Wolf, without ever having to beat it on Expert difficulty.