The Regional Rail Link, Victoria's biggest public transport project since the City Loop was built, has been hit by a two-month delay and will not open until late June.

Trains were scheduled to begin operating along the new 45-kilometre link between Southern Cross Station and West Werribee in April, but will not do so until June 21, after the government made a surprise decision to delay the link's opening.

In a statement issued on Thursday evening, the Andrews government said a shortage of available V/Line trains had forced it to defer the grand opening of the $4.3 billion project, the first major expansion of the state's rail network in more than 30 years.

The eight-week delay will also hold back until June wholesale changes that are planned to Melbourne's train, tram and bus timetables.

Labor has blamed the delay on the former Napthine government, accusing it of planning to open the link without any trains to run a full timetable.