An escalating confrontation on Native land southwest of Tucson threatens to upend decades of uneasy cooperation between the Tohono O’odham Nation and the United States.“It’s like me bulldozing right through the Arlington Cemetery,” said Verlon Jose, the chairman of the Tohono O’odham Nation.

The Trump administration wants to build a towering 62-mile barrier that would bisect the Tohono O’odham homeland along the international boundary with Mexico, and construction could begin as soon as October. Leaders of the Native American nation say it will restrict member movement, endanger wildlife, destroy sacred burial sites and undermine tribal sovereignty while doing little to tamp down illegal activity.

The tribe has sued to prevent construction, but the federal government is pushing ahead as it seeks to wall off most of the southern frontier. The Tohono O’odham Nation, which occupies a reservation roughly the size of Connecticut, has for years allowed federal immigration authorities to operate bases and surveillance towers on its land. The tribe spends $3 million a year on border security, and its police force works closely with Border Patrol agents.

“We’ve bent over backwards to work with the federal government,” Mr. Jose said. “But we will never agree to a wall.”