The "whisky train" will make its first winding 200-mile journey this week from a goods yard in Elgin to Grangemouth, where it will be distributed to bottling halls and warehouses across the central belt.
It is the first time there has been a substantial volume of goods, including whisky, transported by train from Elgin since the mid-1980s, with renewed interest in the environmental benefits of rail freight. Several whisky producers are working on the trial, including Diageo, Chivas Brothers, Dewar, Whyte and Mackay and Glen Turner.
For every journey, it is calculated 29 lorry trips will be saved, with the A9 among roads to benefit from a reduction in traffic.
Tony Jarvis, senior development manager for transport at Highlands and Islands Enterprise, said: "This project has the potential to support the expansion of one of Scotland's key exporting industries.
"Any increase in whisky production requires a proportional increase in transport, with additional pressure on roads, and carbon emissions."
Trains will run twice a week with plans to use the return journey for deliveries of other goods such as malt, barley and empty bourbon casks to hold the next batch of spirit.
The trains are being brought on track by the Lifting the Spirit project which, over two months, will be analysed by an academic to determine its long-term potential.
Richard Lochhead, Cabinet Secretary for Rural Affairs and the Environment said: "The area covered by this trial is home to 77 distilleries which produce 85% of all of Scotch malt whisky. That equates to a lot of freight on Scotland's roads.
"The Scottish Government is keen to see more goods moved by rail or water, where this is commercially viable, to ease traffic congestion and help the environment."
The development comes after calls from the Rail Freight Group to upgrade the railway line between Perth and Inverness to ease travel on the A9 trunk road.
Julie Hesketh-Laird, director of operational and technical affairs at the Scotch Whisky Association, said: "The Scotch whisky industry continues to grow. Ongoing investment by producers is allowing the industry to expand to meet global demand for Scotch."