Alexis Lopez said he had resigned a week ago as secretary of the National Defense Council, an advisory body to the government, but was making the decision public on Wednesday for the first time.
His departure adds to signs of increasing pressure on socialist President Nicolas Maduro, who has been resisting months of street protests by opponents demanding elections to remove him from office.
Analysts say Maduro relies on the support of the military high command to stay in power.
The most prominent is Attorney General Luisa Ortega, who has tried to sue the government for breaches of human rights and democracy.
Like Ortega, Lopez said he was acting in protest at Maduro's proposal to set up an elected assembly to rewrite the constitution in response to the protests.
Maduro's opponents say the assembly is a ploy to cling to power. They blame him for an economic crisis that has caused desperate shortages of food and medicine in the oil-rich country.
Lopez said in a statement he was quitting "due to my disagreement with the way of proceeding to summon a national constituent assembly" without letting Venezuelans vote on the reforms in a referendum.
He expressed his "esteem" for Maduro, saying the president had offered him an alternative post that he had turned down.
Some analysts have said divisions within the government camp could strengthen the opposition's hand in trying to remove him.
Rocio San Miguel, a military affairs specialist of the civil association Citizen Control, however played down the impact of Lopez's move.
Venezuela's defense minister and commander of the armed forces, Vladimir Padrino, reiterated his support for Maduro's constitutional plan on Tuesday.
Last week, Padrino had sounded a note of moderation by warning security forces not to rob and assault protesters.
State prosecutors say 68 people have been killed so far since a wave of protests against Maduro erupted on April 1.
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