The military fired the target, a Scud-like missile with a range of a few hundred miles, from a decommissioned amphibious assault ship near Hawaii's island of Kauai.
The USS Lake Erie, based at Pearl Harbor, fired two interceptor missiles that shot down the target in its final seconds of flight about 12 miles above the Pacific Ocean.
The target was shot down about 100 miles northwest of Kauai about five minutes after it was fired.
The $40 million test showed Navy ships are capable of shooting down short-range targets in their last phase of flight using modified missiles the service already has, the military said.
The Navy and the Missile Defense Agency have already demonstrated that ships equipped with Aegis ballistic missile defense technology can intercept mid-range targets in midcourse of flight.
The Lake Erie in February shot down a U.S. spy satellite that had lost power and become uncontrollable. Military commanders worried the satellite would break up and spread debris over several hundred miles if it fell to Earth on its own.
The shootdown was the Aegis ballistic missile defense program's first real-world mission.
Rear Adm. Brad Hicks, the program's director, told reporters in a conference call after Thursday's test that the Lake Erie fired two interceptors to increase the probability of interception.
The Navy does that when a target is close to hitting the surface, he said.
Over the next 20 months, the military plans to install terminal-phase missile interception capability on all 18 Navy ships equipped with Aegis ballistic missile defenses, Hicks said.
He said the technology would give commanders more options to defend against missiles, particularly if the Patriot missile defense system a land-based technology designed to shoot down missiles in their final phase of flight was unavailable.
"If I don't have a Patriot nearby on a shore station to do a short-range threat, near the defended area, I have nothing," Hicks said. "The flexibility of having a ship to complement the Patriot, or to be there when it can't be, is very high on a warfighter priority."
In the last Aegis missile defense test, in November, the Lake Erie fired two interceptors to destroy two ballistic missile targets simultaneously in space.
That marked the first time the U.S. missile defense system shot down two ballistic missiles at once in space.
In December, a Japanese naval vessel equipped with the Aegis ballistic missile defense system shot down a missile target off Hawaii. Japan became the first U.S. ally to intercept a missile from a ship at sea in that test.