It called in a statement on "all political actors, and in particular the government, to ensure full respect for human rights and fundamental freedoms and the existing constitution".
And it urged them to "prevent or suspend ongoing initiatives such as the new Constituent Assembly which, instead of fostering reconciliation and peace, foment a climate of tension".
Maduro was due to install the new assembly of his allies today, dismissing an international outcry and opposition protests saying he is burying democracy in his country.
That has raised fears of violence that could add to a death toll that has risen to more than 125 over the past four months.
"The Holy See appeals firmly to all of society to avoid all forms of violence and invites, in particular, the security forces to refrain from excessive and disproportionate use of force," the Vatican said today.
The Vatican's appeal was its first official reaction following the vote last weekend and its silence had not gone unnoticed.
Pope Francis had offered in April to intervene as a mediator to try to end the deadly political crisis, but to no avail.
Today the Vatican urged Venezuela to abide by conditions it outlined in December: the establishment of an electoral calendar, the freeing of opposition supporters held in prison, allowing foreign health aid and giving back parliament its powers.
Forty countries have admonished Maduro for seeing through the creation of the new assembly, with the US hitting him with sanctions this week and US President Donald Trump branding him a dictator.
"In 30 years of Vatican diplomacy I have never seen anything like this, which means that the Vatican considers the situation to have reached an extreme limit," he told AFP.
Earlier this week Secretary of State Pietro Parolin, the former apostolic nuncio in Venezuela, denied the Vatican's attempt at mediation was "a failure".
"There's no failure. The Holy See's diplomacy is a diplomacy of peace," he said in an interview with the Avvenire daily.
"In the case of Venezuela there can be differing opinions but the important thing is to try to find feasible solutions based on the situation, particularly bearing in mind the conditions on the ground," he added.
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