A protest group is to appeal against a High Court ruling that effectively gave the go ahead to the London-Birmingham section of the HS2 high-speed railway.
The government claimed a "landmark victory" in March when it won nine out of 10 points challenging the scheme.
But an appeal from the HS2 Action Alliance (HS2AA), concerning the environmental impact, will be heard by the Court of Appeal on June 10.
The government said it will "continue to defend any challenge" in the courts.
HS2AA, which represents more than 70 affiliated groups and residents' associations and a golf club, claims there were failures in the consultation process and in assessing the high-speed link's environmental impact.
Its claim was one of the nine points brought by protesters that was thrown out in the March ruling by Mr Justice Ousely.
The sole successful legal challenge concerned the way in which the property compensation consultation for HS2 had been carried out. This part of the consultation is now being re-run.
It concerned the £16.3bn first phase of the rail project.
HS2AA has raised the necessary £100,000 to appeal against the High Court's ruling.
Its director Hilary Wharf said today: "The British public have joined the NAO and Major Projects Authority and are putting their hard-earned cash up to hold the government to account for their incompetent handling of this white elephant."
Supporters of HS2 says it holds massive future benefits for the economy but critics have suggested these have been over-estimated.
A Department for Transport spokesman said: "The judge was firm in dismissing these challenges and the government will continue to defend any challenge in the Court of Appeal.
"It is unfortunate but inevitable that opponents of HS2 will do what ever they can to delay the government's plans, but the government remains committed to delivering HS2 as quickly as possible."
The court challenge comes after the Cabinet Office published its Major Projects Authority's annual report which showed ministers' assessments of high-profile schemes.
The HS2 project was one of 23 schemes to be given an 'amber/red' status indicating ministers had their doubts.
A recent National Audit Office (NAO) report has also questioned the business case and the funding for HS2.