Unlike other catfish which are scavengers, flatheads prey only on live fish. Young flathead catfish feed mostly on invertebrates such as worms, insects and crayfish. When 10 inches or larger, their diet consists entirely of fish-shad, carp, suckers, sunfish, largemouth bass and other catfish (including their own kind). Flathead catfish are eaten by alligators, water snakes, turtles, larger fish, and humans. They reach sexual maturity between the third and sixth year. Spawning season is from late May through August, when the water temperature is between 75° and 80° F.

Males select hollow logs, caves or areas beneath the banks for their nest sites. Males may even improve their selected sites by creating shallow depressions for the females to lay their eggs. Egg number varies greatly depending on female size, but the average is up to 100,000 eggs at a time. Scientists estimate that a female will lay 1200 eggs for every pound she weighs. A female flathead that weights 50 pounds might release 60,000 eggs at a time. After an incubation period of four to six days, the fry (very young fish) will school together at the nest for several days after hatching; afterwards they will seek shelter beneath rocks, roots and other cover and begin their independent lives. Average lifespan of the flathead catfish is 12 to 14 years, but one recorded flathead catfish lived 24 years.

Adults are usually solitary, each staking out a favorite spot under a tree or in a cove, in deep water. At night, they move into shallow areas to feed. Males defend their nest and eggs aggressively. They will fan the nest with their tails to keep the eggs clean and provide them with oxygenated water. If females have been eating poorly, their bodies may conserve resources by not releasing eggs. Poor overall health and certain environmental conditions such as drought or flood can reduce flatheads' ability to spawn. In healthy times, clutches can reach 100,000 eggs, but only a small number will survive.