The incident triggered an investigation and the train was cancelled, forcing passengers to wait one hour for the next service.
News of the safety breach came as conductors were due to stage a second 24-hour walkout tomorrow, with further stoppages planned for Saturday and Sunday.
The manager was standing in for striking conductors on the 11:28am train from Tweedbank to Edinburgh.
He signalled to the driver for the train to depart, but the driver did not proceed because the signal was red, and reported the incident.
An industry source told The Scotsman: “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.
“The driver reported the incident, thus leading to the train being cancelled. An inquiry is ongoing.”
ScotRail confirmed a conductor working a Tweedbank–Edinburgh service gave an “incorrect signal to depart” to the driver.
It said the driver did not accept the signal and the train did not move away. The train was cancelled, which is normal practice in such incidents, and passengers were transferred to the next service.
The manager was taken off duty, following usual procedures.
A ScotRail spokesman said: “Following normal procedure, this was immediately reported to Control and our normal internal investigations have commenced.
“These incidents happen occasionally. There is an agreed re-training and support programme for train crew following operational errors.”