Former attorney-general Jesus Murillo was arrested at his Mexico City home on charges of forced disappearance, torture and obstruction of justice over the abduction and disappearance of the student-teachers in the southwest state of Guerrero.
Murillo was taken to an office of the attorney-general and will be moved to a Mexico City jail, authorities said.
A judge has released 83 more arrest orders - for soldiers, police, Guerrero officials and gang members - in relation to the case, the attorney-general's office said.
During Murillo's 2012-2015 term under then-president Enrique Pena Nieto, he oversaw the highly criticised investigation into the September 26, 2014, disappearance of the students from the Ayotzinapa Rural Teachers' College.
The remains of only three students were ever found and identified, and questions have haunted Mexico ever since.
International experts criticised the official inquiry as riddled with errors and abuses, including the torture of witnesses.
Current President Andres Manuel Lopez Obrador took office in 2018 vowing to clear up what had happened.
Lopez Obrador's administration has tried since 2020 to arrest another former top official, Tomas Zeron, including asking Israel last year to extradite him.
When asked about the government's move to scrutinise the past investigation, Murillo said he was pleased and was open to being questioned, local media reported in 2020.
The attorney-general's office said Murillo co-operated "without resistance".
The arrest comes a day after Mexico's top human rights official, Alejandro Encinas, called the disappearances a "state crime" with involvement from local, state and federal officials.
"What happened? A forced disappearance of the boys that night by government authorities and criminal groups," Encinas told a news conference.
The highest levels of Pena Nieto's administration orchestrated a cover-up, Encinas said, including altering crime scenes and hiding links between authorities and criminals.
Murillo took over the Ayotzinapa case in 2014 and dubbed the government's findings the "historical truth".
According to that version, a local drug gang mistook the students for members of a rival group, killed them, incinerated their bodies in a dump and tipped the remains into a river.
A panel of international experts picked holes in the account, and the United Nations denounced arbitrary detentions and torture during the inquiry.
The "historical truth" eventually became synonymous with the perception of corruption and impunity under Pena Nieto as anger mounted over the lack of answers.
Murillo, who had previously been a federal MP and the Hidalgo state governor, resigned in 2015 as criticism mounted over his handling of the case.
