Charon appears in the Game Over sequence to ferry Flynn across the River Styx. However, this time he will offer to ferry Flynn back to the world of the living. His reason for doing this is that he is overworked, with countless souls waiting to cross, so he wants to ease his load. Since he could set a dangerous precedent by misusing his power as ferryman in this way, a sufficient bribe of Macca (or accumulated Play Coins on the 3DS) is required.
If Flynn chooses to wait in line instead of reviving, an annoyed Charon will tell him that he'll be waiting until after the mountains have turned to dust. Should Flynn insist, Charon will give him a number and dump him nearby to wait. In addition, if Flynn does not have enough macca to revive, Charon will put it on tab and collect it later once enough has been accumulated. Should he perish again before the tab is paid off, Charon will just tell his attendants to toss Flynn into a nearby mountain of souls.
His assistants, two nameless guards, issue the DLC Challenge Quests, Experience of the Afterlife, Money Makes the Underworld Turn, and Death Has Its Applications. They do this mostly to serve Charon, as he's either annoyed by the increased workload, by the consistent poverty of recent arrivals, or vexed because of their ignorance. In either case, the rewards for all three Quests are issued mostly to give Charon some breathing room by easing his workload.
Charon appears before the protagonist after Sid Davis kills him, telling him it's his time to cross the river. However, the protagonist is brought back to life before this can happen. Charon appears in the Game Over sequence to take him across the river after the first time failed.