Monday marks the second day of strike action after a similar walk-off was called on Sunday by the southern European country's largest trade union, the Italian Confederation of Labour (CGIL).
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Thousands of workers and students across Italy joined a general strike and widespread demonstrations on Monday in solidarity with the Palestinians living in Gaza.
Italy’s grassroots unions called for a 24-hour general strike in both public and private sectors, predominantly affecting public transport, trains, schools and ports.
The strike caused disruptions across the country, with long delays for trains and limited public transport in major cities, including Rome and Milan.
The transit of goods was slowed or partially blocked by workers' sit-ins and rallies in Italy’s main ports of Genoa and Livorno.
It's the second day of strike action after a similar walk-off was called on Sunday by Italy's largest trade union, the Italian Confederation of Labour (CGIL).
"The strike is taking place in response to the ongoing genocide in the Gaza Strip, the blockade of humanitarian aid by the Israeli army and the threats against the international Global Sumud Flotilla mission," a statement from the USB trade union federation said.
"USB also denounces the inertia of the Italian government and the European Union, which refuse to impose sanctions on the State of Israel and continue to maintain economic and institutional relations despite the gravity of the situation."
Organisers of the rally in Rome say they expect around 100,000 people to attend.
However, city authorities put the number at 20,000 gathered in front of the Termini central train station around midday Monday, adding that the number was not final and that it was rising.
The Italian government headed by Premier Giorgia Meloni has more recently adopted a harsher tone on its policies as domestic pressure has mounted over the ongoing Israel-Hamas war.
Italy has not joined the European countries including France and the UK that will formally recognise a Palestinian state at this week’s UN General Assembly in New York.