The children were travelling in a large passenger van from Avoyelles Parish, Louisiana, on Thursday, according to Florida Highway Patrol Lieutenant Patrick Riordan.
A lorry and a passenger vehicle collided in the northbound lanes of Interstate 75 and continued across the central reservation and into the path of another HGV and the church van, investigators said.
"Once those semis struck, they both caught fire," Lt Riordan said during a news conference on Friday in Alachua, south of Gainesville.
He said a fifth vehicle came through and either struck people who had been thrown from the wreckage or debris. About 50 gallons of diesel spilled, worsening the fire that also damaged the road in spots.
At least eight people were taken hospital with injuries.
The Louisiana district office for the United Pentecostal Church said on Facebook that Karen Descant, wife of Pastor Descant, suffered multiple injuries. A pregnant woman was stabilised and another women suffered non-serious injuries. Four other children "all have injuries, but the medical staff feel that all of them will heal and recover."
Kevin Cox, superintendent of the church district told CNN: "Please pray for the families who lost precious children, the other accident victims, all involved in the rescue efforts, and the church family. They will need our prayers in the coming weeks and months."
The aftermath closed part of the road in both directions, causing massive delays along the busy north-south corridor.
Debris, including personal property and vehicle parts, was scattered across the road, Florida Highway Patrol said. A helicopter helped search for any victims who may have been in nearby woods.
Nicole Towarek was travelling northbound with her family when they came across the scene. She told the Gainesville Sun that black smoke billowed, people were laid out near vehicles, there were long skid marks across the road and emergency workers were converging on the area.
"We kept seeing these little explosions and fire," she said. "The heat, it was insane."
It was the worst accident on I75 in Alachua County since January 2012, when 11 people died in a chain reaction crash attributed to heavy fog and smoke on the road, which crosses Paynes Prairie Preserve State Park.
Officials were criticised then for not closing the road due to worsening conditions, and later installed cameras, sensors and large electronic signs to help prevent similar crashes.
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