The operator said more than 100 services were cancelled on Wednesday.
ScotRail will move to a temporary timetable in January in a bid to offset the impact of the new Omicron variant.
The data emerged as First Minister Nicola Sturgeon confirmed a record 15,849 people tested positive in Scotland on Tuesday.
ScotRail communications director David Ross said 320 staff were currently absent, including more than 100 drivers and about 60 conductors.
It is about about 6% of the workforce of 5,300 drivers, conductors and engineers.
The firm has repeatedly warned that staff absences were affecting services over the last couple of weeks, asking passengers to check services before they travel.
Mr Ross told BBC Radio Scotland's Drivetime programme that the 10-day isolation rule on its own was not to blame for the cancellations as it only accounted for up to 25% of staff who are currently absent.
He added: "The most significant impact is people who have tested positive themselves or who have symptoms and are awaiting a PCR result so, regardless of the household contact policy there will still be an impact on our services."
Mr Ross said fewer people are using trains at the moment but urged passengers to check before they travel, space out as much as possible and to wait for the next service if a train is too busy.
ScotRail has said it will continue to review staff absence levels over the coming weeks in the event of a further spike in cases.
Routes affected by the timetable revision, which starts on Tuesday, include:
* Glasgow Central - Neilston / Newton
* Glasgow Central - East Kilbride
* Glasgow Central - Lanark
* Glasgow Central - Ayrshire / Inverclyde / Stranraer
* Glasgow Central - Edinburgh Waverley via Shotts
* Glasgow Queen Street - Edinburgh Waverley via Airdrie & Bathgate
* Glasgow Queen Street - Edinburgh Waverley via Cumbernauld & Falkirk Grahamston
* Glasgow Queen Street / Edinburgh Waverley - Stirling / Dunblane / Alloa
* Glasgow Queen Street - Anniesland via Maryhill
* Edinburgh Waverley - Dunbar / North Berwick
* Edinburgh Waverley - Fife
* Edinburgh Waverley - Newcraighall / Tweedbank
A full list of the changes can be found on the ScotRail website.