The guided missile was fired into the East Sea (Sea of Japan) on Sunday afternoon, a defence ministry spokesman told AFP without elaborating.
On Saturday the North fired three short-range missiles off its east coast, apparently as part of a military drill.
The North's short-range missile launches are not unusual but come at a time of heightened alert on the peninsula, following Pyongyang's February nuclear test which sparked tougher UN sanctions.
Angered by the sanctions and by a joint US-South Korean military exercise, the North for weeks threatened nuclear or conventional attacks on Seoul and Washington.
The South and its US ally had earlier been watching for any test by the North of medium-range Musudan missiles. But a US defence official said early in May the two mid-range missiles had been moved from their launch site.
However South Korea's unification ministry, which handles cross-border relations, said the short-range launches also pose threats to the region and should be stopped immediately.
"We find it deplorable that the North does not stop provocative actions such as the launch of guided missiles yesterday," said unification ministry spokesman Kim Hyung-Seok, before the latest exercise.
"We call on the North to take responsible actions for our sake and for the sake of the international community."
UN chief Ban Ki-moon, speaking in Moscow, also called for Pyongyang to "refrain from" further missile tests. He said it was time for it to resume talks with the international community and reduce tensions.
The US State Department urged Pyongyang to exercise restraint, without specifically commenting on the launches.
It was unclear what type of missiles were fired Saturday and Sunday.