The aircraft's co-pilot took control of the plane after the captain became incapacitated, and landed safely at Syracuse Hancock International Airport shortly after 7am.
The plane, an Airbus A320, was en route from Phoenix to Boston when it was diverted to Syracuse in response to the medical emergency.
"Unfortunately, our pilot passed away," American Airlines said in a statement. "We are incredibly saddened by this event, and we are focused on caring for our pilot's family and colleagues."
The airline and federal aviation officials declined to release the name of the pilot or any details about the source of the illness.
While rare, it is not unheard-of for a pilot to suffer a medical emergency and die during a flight.
In 2009, a Continental Airlines jetliner carrying 247 passengers landed safely at Newark Liberty International Airport after the 60-year-old captain died of a heart attack.
The plane was on autopilot when he died. His co-pilots quickly seized the controls and landed the plane safely.
Only two months before that episode, a passenger landed a privately operated twin-engine plane at Southwest Florida International Airport in Fort Myers when the pilot died after takeoff. The passenger, who had training as a pilot, saved four lives.
In January 2007, a pilot of a Continental 757 bound from Houston to Puerto Vallarta, Mexico, with 210 passengers onboard died after takeoff. The flight landed safely after being diverted to McAllen-Miller International Airport in Texas.
A spokeswoman for American Airlines could not be sure if a pilot for the airline had ever died in flight, but said there were clear procedures to follow in the event of emergencies.
If it is evident that the captain of an airliner has become incapacitated, the first officer is expected to take control of the aircraft and then seek help.