Pope Francis made a last-minute call for the assembly to be suspended on Friday.
A statement issued by the Vatican urged Maduro to suspend initiatives such as the new constituent assembly, which, rather than fostering "reconciliation and peace", encourages a climate of "tension and confrontation", as well as putting the future of the country at stake.
Venezuela's ruling party have installed a new super assembly that supporters promise will bring peace to the country and critics fear will be a tool for imposing absolute rule.
The installation of the all-powerful constitutional assembly is likely to intensify a political crisis that has brought four months of protests that left at least 120 people dead and hundreds jailed.
Maduro vows the assembly will strip opposition politicians of their constitutional immunity from prosecution, while members of congress say they will only be removed by force.
But the opposition is struggling to regain its footing in the face of the government's strong-armed tactics and the re-emergence of old, internal divisions.
The Vatican statement expressed profound concern for the radicalisation and worsening of the crisis, including the increase in deaths, injuries and arrests of protesters.
It called on all the country's government, to guarantee full respect for human rights and basic freedoms, as well as for the existing Constitution.
The statement also called for a negotiated solution that takes into account "the grave sufferings of the population due to the difficulty of obtaining food and medicine, and the lack of security".
Pope Francis asked for all people in Venezuela, in particular the security forces, to avoid violence or an excessive use of force and added that he will constantly pray for the country at its moment of crisis and hoped others will too.