Energy Resources Australia (ERA) had applied for a 10-year lease renewal on the Jabiluka uranium mine, which the company previously held for 42 years but was knocked back on Friday.
Mining minister Mark Monaghan said the decision to not renew was based partially on advice from the federal government.
"We have gone through a thorough process to ensure that all stakeholder views have been considered in this decision," he said.
"The Federal Government advice, along with the wishes of the Mirarr people, were critical to this process and outcome."
The Northern Territory government declared special reserve status over the Jabiluka area, which is in the surrounds of Kakadu National Park, in May.
This prevents any future applications for the grant of a mineral title over the Jabiluka area once the current lease ceases on August 11.
The Australian Conservation Foundation welcomed the decision, calling it a "huge win" for traditional owners.
"This decision allows a line to be drawn under the divisive era of uranium mining in Kakadu," a statement read.
"This is a responsible decision that ends the threat that has hovered over this very special place for four decades."
Mirarr people have long opposed any mining in the area, holding protests in the late 1990s and early 2000s when more than 5000 people travelled to Kakadu to prevent uranium mining at Jabiluka.
Energy Resources of Australia, a subsidiary of the Rio Tinto Group, has been contacted for comment.
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