The three men, Robert Nowlin, Paul Holm, and Joel Quiles, have all been suspended without pay and could face prison time.
They were arraigned Friday on grand larceny and criminal possession of stolen property charges in Nassau County District Court.
Authorities say the workers removed wire cables from job sites, loaded them onto LIRR trucks and then transferred them onto their own vehicles. They say wire was later sold to local recyclers for cash.
Authorities say the thefts occurred between February and April 2011 and December 2012.
All three pleaded not guilty. They have been suspended without pay from the railroad.
The arrests come after 15 other LIRR employees were convicted of similar crimes.
"Stealing material that belongs to the public is not only a violation of the public's trust, but an act of stealing from, in this case, every rider on the Long Island Rail Road," Nassau County DA Kathleen Rice said. "I'm proud of the cooperation between my office, the MTA Inspector General and the MTA Police Department to hold public employees accountable to the people they serve."
"The LIRR does not tolerate employees who steal from their employer and from the taxpayers who support our operation," said LIRR President Helena E. Williams.
The Associated Press contributed to this story.