A Scotrail manager gave the all-clear for a train to travel through a red signal during Tuesday's RMT strike - leading to the service being cancelled.
The incident sparked a major investigation and after the train was cancelled passengers on the new Borders Railway had to wait an hour for the next one.
News of the safety breach came as conductors staged a second 24-hour walkout today, with further stoppages planned for Saturday and Sunday.
Read more: Second strike day spells further disruption on the trains
The manager was standing in for striking conductors on the 11.28am train from Tweedbank in the Borders to Edinburgh Waverley.
He signalled to the driver for the train to depart, but the driver did not proceed because the signal was red, and then reported the incident.
A union source said: "The guard gave two bells to the driver which tells the driver to proceed away, but the signal was at danger so it was a direct rule book discrepancy.
Read more: Second strike day spells further disruption on the trains
"The driver reported the incident, thus leading to the train being cancelled. An inquiry is ongoing."
ScotRail confirmed an "incorrect signal to depart" was made to the driver.
It said the driver did not accept the signal and the train did not move.
The manager was taken off after the error, in accordance with rail industry safety procedures.
A Scotrail spokesman said: "Following normal procedure, this was immediately reported to control and our normal internal investigations have commenced.
Read more: Second strike day spells further disruption on the trains
"These incidents happen occasionally. There is an agreed re-training and support programme for train crew following operational errors."
Around seven in 10 trains were scheduled to run during yesterday's (THURS) strike, but a number were cancelled with replacement buses available on some routes.
A ScotRail spokeswoman said: "We are now working on Saturday's train and bus strike timetables, which should be broadly similar to Tuesday and Thursday's."
The bitter dispute is over plans to change conductors' duties in a new fleet of electric trains being introduced across the Central Belt from next year.
Drivers would take over control of doors and passenger safety, with conductors left to check tickets.
Lower-paid ticket examiners would perform that role on some services.
Mick Cash, general secretary of the RMT, said: "RMT has warned repeatedly that Scotrail's scabbing operation represents a massive gamble with passenger and staff safety.
"They have crashed on regardless of those warnings and the consequences are wholly Abellio/Scotrail's responsibility.
"Instead of sending ill-trained and ill-advised managers out to try and do the safety critical job of the guard, the company should be putting their time and effort into negotiating a settlement to this dispute."
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