Spain's King Felipe VI said Catalan leaders have broken the laws of the state and showed an "inadmissable lack of loyalty."
He called their behavior "irresponsible," saying it risked the country's economic stability.
The king's comments come as tens of thousands of people took to the streets of Catalonia and road traffic, public transport and business were disrupted on Tuesday in protest against Sunday's violent crackdown by Spanish police on an outlawed independence referendum.
Catalan leader Carles Puigdemont has said the referendum is valid and its result must be implemented. Spain's Constitutional Court prohibited the ballot, siding with Madrid which argued that it contravened the country's 1978 constitution which bars breaking up the country.
The referendum has plunged Spain into its worst constitutional crisis in decades, and is a political test for Rajoy, a conservative who has taken a hardline stance on the issue. Outside of Catalonia, Spaniards mostly hold strong views against its independence drive.
Catalonia, Spain's richest region, has its own language and culture and a political movement for secession that has strengthened in recent years.
Pro-independence parties who control the regional government staged Sunday's referendum in defiance of Spanish courts that had ruled it illegal. Some 900 people were injured on polling day when police fired rubber bullets and charged at crowds with truncheons to disrupt the vote.
Those who participated voted overwhelmingly for independence, a result that was expected since residents who favour remaining part of Spain mainly boycotted the vote.
Opinion polls conducted before the vote suggested only a minority of around 40 percent of residents in the region back independence. But a majority want a referendum to be held, and protesters said the violent police crackdown against the ballot had energized the secessionist camp.