The calls, a day after high-level government talks between the two sides were canceled, were made at 9 a.m. and 4 p.m. by the liaison officer in the South Korean Ministry of Unification but no one responded from the other side, Yonhap News quoted officials as saying.
The telephone line, running through the Panmunjom truce village, had recently been reopened to enable communication between the two sides.
Yonhap, quoting a ministry source, said Wednesday's development may indicate the North had decided to again cut off the line, which would leave the South no other direct link to the other side.
Three military hotlines set up in the past were disconnected, with growing tensions arising from the North's long-range missiles and its third nuclear test.
The high-level talks aimed at easing tensions were called off Tuesday, a day before they were to begin, because of disagreement over the status of the delegation leaders.
The talks would have dealt with various issues, including resumption of operations at the inter-Korean industrial complex in the North's border town of Kaesong. North Korea suspended the Kaesong operations in April amid rising tensions.