Following a July coup, the West African country said in mid-March that the 2012 cooperation agreement had been "unilaterally imposed" by the United States.
Students and several prominent figures from the military regime were amongst the crowd in front of the National Assembly Headquarters in Niamey.
The crowd was heard chanting "Down with American imperialism" and "The people's liberation is on the march".
French troops were expelled at the end of 2023, but about 1,000 American soldiers remain based in Agadez city in the north.
In late March, Niger said the US would submit a proposal to "disengage" its soldiers from the country. Washington declined to comment, but said it contacted Niger to "obtain clarification".
"They said they (the Americans) were going to leave, so let them leave in peace and quickly," shouted Sheikh Ahmadou Mamoudou, a well-known religious leader.
Flags from Burkina Faso, Mali, Niger and Russia were visible but organisers asked demonstrators to refrain from slogans insulting the US or burning its flags.
In March Niger joined neighbours Mali and Burkina Faso in the creation of a joint force to battle the long-running jihadist rebellions raging in the three nations.
The three countries have turned their backs on former coloniser France and strengthened their ties with Russia.
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