In a statement, he said he no longer spoke on behalf of the movement.
He added he was stepping down because of "internal changes" within the 20-year-old, far-left guerrilla group, and denied rumours that he was unwell.
The group has been fighting for greater recognition of the rights of indigenous people in the state of Chiapas.
"I declare that the one known as Insurgent Subcomandante Marcos no longer exists," Rafael Guillen Vicente, better known by his nom-de-guerre Subcomandante Marcos, said on a Zapatista website.
"The voice of the Zapatista National Liberation Army (EZLN) will no longer come from my voice," he added.
For some time there have been rumours that he was in ill health, but he rejected those out of hand, saying that such reports had been spread by the rebel army for their own benefit.
'Unexpected'
His announcement comes just a day after he was seen in public for the first time in many months, when the enigmatic masked rebel attended a memorial for another key Zapatista leader in Chiapas, one of the poorest regions of Mexico.
The BBC's Will Grant in Mexico City says there appeared to be little outward sign that the rebel leader was about to retire from public life.
Subcomandante Marcos has reinvented himself in the past, launching himself as an alternative presidential candidate one year.
But it seems likely his time at the forefront of an organisation which once rocked the Mexican political establishment to its core has come to an end, our correspondent adds.
Subcomandante Marcos led an armed uprising in Chiapas on New Year's Day 1994.
The rebellion sparked several days of sustained fighting with the federal government, leaving dozens of people dead.
A peace pact was later signed but the Zapatistas' demands were never met and they created their own autonomous justice, health and education systems in several communities.
The head of the Zapatista rebels in southern Mexico, known as Subcomandante Marcos, has announced that he is leaving the group's leadership.