Illinois State Police said the dead passenger was a woman who had been seated in the front-upper section of the bus, above the driver. More than 30 people were transported to area hospitals.
Authorities told ABC News that they believed the cause of the wreck was a blown tire.
The bus, which had 64 passengers listed on its manifest, according to the state police, slammed into a concrete bridge support on Interstate 55 Thursday afternoon near the town of Litchfield, Ill. The company said it was headed to St. Louis and then Kansas City.
Police said they expected the highway to remain closed until late Thursday evening and possibly into Friday morning.
Thirty ambulances, five helicopters and seven fire departments were sent to the scene, the state police said.
A spokesperson for St. Francis Hospital in Litchfield told ABC News that it had received 21 patients.
Three other passengers were taken to Staunton Hospital and another three to Hillsboro Hospital. In addition, some passengers were taken to Memorial Hospital in Springfield.
The bus, according to a Megabus spokesperson, was traveling along a route between Chicago, St. Louis, Columbia and Kansas City. Litchfield, the site of the wreck, was about 55 miles north of St. Louis.
The National Transportation Safety Board said it was monitoring the situation, but had yet to make a decision on whether to launch a full investigation.
In a statement, Megabus said: "Safety remains our number one priority. The thoughts and prayers of our entire staff go out to the passengers involved. Megabus.com will continue to provide more details as they become available."
Operating more than 250 buses in 80 cities, Megabus is one of the largest "curbside" bus operators in the U.S. It picks up passengers on the street, not at a bus depot, and offers Internet fares as low as $1.
In September 2010, four passengers died after the driver of a Toronto-bound Megabus missed an exit and crashed near Syracuse, N.Y.
Megabus says that it has the highest safety rating from the federal government, but concern is growing over the overall safety of curbside bus operators.
According to the NTSB, the fatal accident rate for curbside bus operators is seven times that of Greyhound.
In May, after a rash of deadly accidents, the Department of Transportation launched surprise inspections in 13 states and the District of Columbia. That crackdown led to 26 bus companies being shut down.
ABC News' Wendy Fisher, George Sanchez, Andrew Evans and Lisa Stark contributed to this report.