The regional government of Castilla La Mancha confirmed the death toll and said another 58 people were receiving medical treatment at hospitals.
Rescue workers searched the wreckage through the day for survivors and bodies.
The collision came just nine months after another deadly crash involving high-speed trains operated by the same two companies in southern Spain in January.
"Today all of Spain is grieving," Spanish Prime Minister Pedro Sanchez said from the crash site, where he announced that his government would declare three days of national mourning.
Sanchez called for increased safety measures to restore confidence in a high-speed rail system that was once a source of national pride.
"It is unacceptable for a country like Spain to suffer two high-speed rail accidents in the same year," said Sanchez, who faces a national election on November 29.
Spain's rail system operator ADIF said the crash occurred on Wednesday morning in the central province of Toledo, involving two high-speed trains carrying hundreds of passengers and heading south in the same direction from Madrid to Seville.
A train operated by the state-owned RENFE company struck the rear of another train operated by private company IRYO that was stopped.
The collision derailed two carriages on the tail of the IRYO train and the front of the RENFE train.
Rail service was suspended between Madrid, Ciudad Real and Andalusia.
Transport Minister Oscar Puente said the crash was caused by an "inexplicable" order from a central command centre for Spain's trains after a security system had failed.
He said the train engineers were not to blame.
Puente said that at 8.30am the security system on a stretch of 63km stopped working due to a computer malfunction, causing all trains on that stretch to stop.
But the central command told the RENFE train to start running again without any speed restrictions, and it ended up colliding with the stopped IRYO train at 8.53am.
Puente said the train should never have been told to start moving again since there was still a "stop signal" active on that line.
And if it had, at least it should have been told to go at reduced speed.
Authorities were still studying information to see how fast the train was going, but it could have been at 200km/h for that stretch of the track, Puente said.
The crash, which occurred in a flat stretch surrounded by olive fields in between the towns of Urda and Los Yebenes, left carriages shredded into bits of metal and other material, scattering debris down a gravel slope.
Authorities said 397 passengers were in the RENFE train and 147 passengers in the IRYO train.
Spain's King Felipe VI also visited the crash site.
"We share in the grief of the families and loved ones of those who lost their lives, and we send our heartfelt support to the injured and all those affected," Spain's royals said in a statement.
Spain's soccer federation postponed all 24 Copa del Rey games scheduled on Wednesday.
The wreckage of Wednesday's crash reminded many of the aftermath of another train collision in January, when 46 people died in southern Spain when an IRYO train jumped the track and hit an oncoming RENFE train.
That sent the first two cars of the impacted train plummeting down a four-metre slope.
That crash was the deadliest in Spain since a 2013 crash that killed 80 people.
Before 2026, there had been no fatal crash on Spain's dedicated high-speed lines since the network opened in 1992.
