Mexican security forces surrounded the Tijuana entrance of the tunnel.
The tunnel was discovered on Tuesday and links warehouses in Tijuana, Mexico, with San Diego.
"It is clearly the most sophisticated tunnel that we have found in the last five years, perhaps ever," a US police spokeswoman in San Diego said.
A similar tunnel was discovered in the area two weeks ago, adding to dozens that have been found in recent years.
Local media describe Otay Mesa as a busy area containing many warehouses, making it easier for smugglers.
The tunnel extends twice as far into the US as it does into Mexico.
The tunnel is 15ft (4.5m) deep and contains both lighting and transport carts, AFP news agency reports.
Mexican security forces found the entrance in a Tijuana warehouse after being alerted to the discovery by the US authorities.
The warehouse is near Tijuana airport, and also stands close to the local headquarters of the federal police.
Mexican security forces seized three tons of marijuana at the tunnel's entrance, which they suspect had only recently been put into use.
The use of underground tunnels for smuggling has increased in recent years as the US authorities have clamped down on overland smuggling activity.