The move angered Russia, which opposes having the advanced military system in its former area of influence.
Nato secretary general Jens Stoltenberg tried to reassure Russia as he spoke at a ceremony attended by US, Nato, and Romanian officials at the Soviet-built base, 180km south-west of Bucharest.
The missile-defence site “in no way undermines or weakens Russia’s strategic nuclear deterrent”, Mr Stoltenberg said at the opening ceremony.
“This site in Romania, as well as the one in Poland, are not directed against Russia.
"The interceptors are too few, and located too far south or too close to Russia, to be able to intercept Russian intercontinental ballistic missiles.”
He said the interceptors were designed, “instead, to tackle the potential threat posed by short and medium-range attacks from outside the Euro-Atlantic area”.
US officials said the Romanian missile shield, which cost $800m, is intended to fend off missile threats from Iran and is not aimed at Russia.
Mr Stoltenberg noted that Moscow had unilaterally terminated co-operative dialogue about missile defence in 2013.