Video from CNN affiliate KTRK-TV showed the school bus on its side, its front smashed, underneath an overpass for Interstate 610's South Loop in southeast Houston.
Witnesses told KRTK that the bus had been on the highway but fell from the overpass. The guardrail on the highway was heavily damaged, the station reported.
The bus was headed to Houston's Furr High School, which also houses REACH Charter High School, when the crash happened at about 7 a.m., the Houston Independent School District said. It wasn't immediately clear which school the students attended, the school district said.
Two female students died, and everyone else aboard -- two students and the driver -- were seriously injured and taken to hospitals, according to the school district. No conditions or names were released.
"We are deeply saddened by this tragedy," district Superintendent Terry Grier said. "I ask all of the HISD community to join me in praying for all of those involved."
Police were investigating the crash, the school district said.
The bus driver has been with the school district for about three years and has a clean driving record, the district said.
The bus last underwent a maintenance inspection in June, according to school officials.