"An EU Commission mission will be at Spain-Gibraltar border on the 25th of September, to check border, free movement of people/goods, including smuggling," Commission spokesman Olivier Bailly said on his Twitter account.
The latest row between London and Madrid over the British outpost broke out after Spain introduced stringent border checks which have led to waits of up to five hours for motorists trying to enter the territory.
Gibraltar has accused Madrid of imposing the checks in retaliation for its decision to place concrete blocks in the sea as part of its efforts to improving fishing.
The Commission in August promised the observer mission to ensure border controls are applied "proportionately", with British Prime Minister David Cameron urging the EU to send the team without delay.
Spain ceded Gibraltar to Britain in perpetuity in 1713 but has long argued that it should be returned to Spanish sovereignty. London says it will not do so against the wishes of Gibraltarians, who are staunchly pro-British.
Cameron last week praised the people of Gibraltar for facing "the pressures of recent months with enormous dignity" and said their relationship with Britain was "solid, sure and enduring".
Spanish Prime Minister Mariano Rajoy earlier this month said he had twice discussed the dispute with Cameron during the recent G20 summit in Russia.
The talks were "useful," Rajoy said, calling for "common sense."