It said footage shown on Globovision implicitly called for Mr Chavez to be killed. The station denies the claim.
Police fired tear gas and plastic bullets as thousands protested across the country against the earlier closure of Venezuela's oldest TV network.
Mr Chavez said Radio Caracas TV (RCTV) had tried to undermine his government.
Communications Minister Willian Lara said Globovision had called for the death of Mr Chavez by airing footage of the 1981 assassination attempt against Pope John Paul II with the song "This Does Not Stop Here" sung by Ruben Blades, now Panama's tourism minister.
"The conclusion of the specialists ... is that (in this segment) they are inciting the assassination of the president of Venezuela," Mr Lara said, as he filed a lawsuit against the news network at the state prosecutor's office.
The government was also suing the US station CNN for allegedly linking Mr Chavez to al-Qaeda, Mr Lara said.
"CNN broadcast a lie which linked President Chavez to violence and murder," he said.
In a statement, CNN said they "strongly deny" being "engaged in a campaign to discredit or attack Venezuela".
Globovision director Alberto Federico Ravell rejected the accusations against his station as "ridiculous".
Globovision was the only TV station to air footage of a large demonstration against the government's growing control over the media.
Meanwhile, protests are continuing across Venezuela after Mr Chavez refused to renew the licence of RCTV.
In one of the largest demonstrations in the capital, Caracas, police fired tear gas and rubber bullets at up to 5,000 protesters, some of whom tossed rocks and bottles at police.
Within seconds of RCTV's closure, the insignia of a new state-sponsored broadcaster, TVES, appeared. Fireworks exploded across Caracas, as Chavez supporters celebrated the end of RCTV.
The president says the new channel will better reflect his socialist revolution but RCTV and rights groups say Mr Chavez is limiting freedom of expression.
Employees of Venezuela's most watched channel embraced and chanted "freedom", before bowing their heads in tearful prayer during their final minutes on air.
Germany, which holds the European Union presidency, expressed concern over Mr Chavez's decision.
Robert Menard, from International media rights group Reporters Without Borders, said the closure was a "major setback to democracy and pluralism".
In a broadcast all of the country's TV stations were obliged to run, Mr Chavez said it had been his decision to shut down the station because it had become "a threat to the country".
RCTV's general manager Marcel Granier said Mr Chavez was acting illegally and described the move as "abusive" and "arbitrary".
RCTV will still be available on cable, but losing its public broadcast frequency will deprive it of most of its audience.
The new state-sponsored channel TVES launched with programmes that Mr Chavez said would better reflect society, including a film about independence hero Simon Bolivar.
The government provided $4m (