The Aquarius, which is run by SOS Mediterranee and Doctors without Borders (MSF), is now on its way to Spain.
The ship had previously tried to dock in Italy, but the country’s new anti-migrant Minister of the Interior, Matteo Salvini, refused to accept it.
Officials in Malta also turned the boat away, claiming the vessel was Italy’s responsibility.
The head of MSF Spain, David Noguera, blamed the EU and the “contradictory policies” of member states for the situation.
And he warned the 10-day trip to Spain and back would inevitably lead to more deaths as migrant boats continue to make the perilous journey to Europe across the Mediterranean from northern Africa.
He said: “The detour of the Aquarius is clearly a symbol of the failure of the European Union and European governments with their contradictory policies.
“It is a very negative precedent. The truth is there is a question on the table as to whether this is a single event which is resolved quickly, or if we will no longer be allowed to dock in Italy and the blockage continues which would completely change rescue operations.”
The Aquarius is carrying 629 migrants from Libya, including seven pregnant women and 123 unaccompanied children.
After rescuing the group from inflatable boats and rafts off the coast of north Africa, the Aquarius headed towards Italy to allow them to disembark.
But Matteo Salvini, Italy’s new Deputy Prime Minister, blocked the ship from docking at any of the country’s ports. The rescue boat is now heading to Spain.
The leader of the far-right League party said he was making good on one of his election pledges by turning the Aquarius away.
He said: “Enough. Saving lives is a duty, transforming Italy in a refugee camp isn’t.
“Italy is done with taking orders, this time there is someone saying no.
“Malta takes in nobody.
“France pushes people back at the border, Spain defends its frontier with weapons.
“From today, Italy will also start to say no to human trafficking, no to the business of illegal immigration.”
But Mr Noguera warned a tough new anti-migrant stance from Italy would ultimately cost lives.
He said: “That the Aquarius was diverted to Spain is not a victory for anyone, not for Salvini although he thinks so, and not for MSF or the European Union and especially not for the refugees.
“Today a boat that should be rescuing people at sea will spend 10 days outside of the rescue zone, and that will cause more deaths, that is obvious.”