Thousands protested in the majority-Muslim region of Ingushetia in October after its veteran leader agreed to a Kremlin-backed border deal with Chechnya.
Activists and clerics have accused Yunus-Bek Yevkurov of surrendering swathes of territory to neighbouring Chechnya and called for his resignation.
A court in Ingushetia ruled in October that the deal was unconstitutional, while the protesters called for a referendum.
In its ruling on Thursday, the Saint Petersburg-based Constitutional Court said the deal was in line with legislation and there was no need for a public vote.
"A referendum in Ingushetia is not necessary for the establishment of borders with the neighbouring republic," court chairman Valery Zorkin said.
Chechnya's Moscow-backed strongman Ramzan Kadyrov praised the ruling, saying he was "grateful" to judges and that the issue had been "put to rest".
But opponents of the land deal were frustrated.
"We had hoped for a miracle," Barakh Chemurziyev, one of the organisers of the protests in Ingushetia, told AFP.
"We wanted to be given an opportunity to conduct a referendum."
He was unsure whether there would now be more protests, saying opponents of the deal needed to "brainstorm" first.
The people of Chechnya and Ingushetia are ethnically close, but tensions have persisted over borders which were arbitrarily drawn up in the early days of the Soviet Union.
Opposition figures said the ruling was the latest evidence that ordinary Russians have no say in how the country is run.
Top Kremlin critic Alexey Navalny said on Twitter that it proved that regional leaders can change borders "as they wish, completely ignoring any laws, opinions and sentiments of the people."
Russia's North Caucasus struggles with a litany of problems including a simmering Islamist insurgency, deep poverty and entrenched corruption.
Since the breakup of the Soviet Union in 1991, Russian troops fought two separatist wars in Chechnya.