He said those in opposition "learn nothing from the past" and said Labour could not claim to want one nation "if you won't back the things that will bring it together".
Concerned with the expense of the project, Labour appear to be cooling towards HS2, which will see the scheme's first phase run from London to Birmingham in 2026 with a second Y-shaped route due to be completed to northern England in 2032/33.
Mr McLoughlin's comments came at a rail conference in Manchester as the Government publishes a strategic business case for HS2, a project estimated to cost £42.6bn, with a further £7.5bn for the trains.
With the strategy document, the Government is also releasing a report prepared by Network Rail and management consultancy Atkins.
This is believed to have concluded that there would have to be 2770 weekend closures on the East Coast, West Coast and Midland main lines if it was to replace the intended capacity of HS2. The study warns that, during a typical weekend, the journey time from London to Leeds could more than double.
Mr McLoughlin said: "Let me say something very direct to those in the opposition who have learnt nothing from the past. You can't say one day you back better infrastructure only the next threaten to stop it being built."