The Sao Paulo metro is the main transport link to the economic capital's World Cup host stadium, which will host the opening ceremony and kick-off match on June 12, and the strike could pose a massive logistical headache for organisers.
Workers decided to strike from midnight Thursday after negotiations on a salary increase fell through. They rejected an offer of 8.7 percent, insisting on at least 10 percent, said the president of their union, Altino Melo dos Prazeres.
The strike will affect 4.5 million daily passengers and could unleash commuter chaos in the sprawling city of 20 million people, which was already hit by a paralyzing bus drivers' strike last month.
Brazil has been gripped by a wave of strikes and protests ahead of the World Cup and elections in October.
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About 12,000 protesters rallied by the Homeless Workers' Movement (MTST), a mainstay of the anti-World Cup protests, marched on Corinthians Arena Wednesday, joining around 400 retired military police and their relatives who were calling for higher pensions.
A year ago, a million protesters flooded the streets during the Confederations Cup, a World Cup dress rehearsal.
The protests turned violent at times, overshadowing the tournament and raising fears of a repeat this year.
The government has also faced criticism for chronic delays and cost overruns.
Workers are still scrambling to finish five of the 12 host stadiums, including Corinthians Arena, which has not had all its seats installed.