The August 5-8 visit by Lee Hee-Ho is ostensibly humanitarian in nature but there has been much speculation about a possible meeting with North Korean leader Kim Jong-Un, who personally approved the trip.
"I hope that this will offer a window for more dialogue, exchange and cooperation," Lee was quoted as saying by her spokesman as she left Seoul on a direct flight to Pyongyang with an 18-member entourage.
The two Koreas technically remain at war and direct flights between the two countries are extremely rare.
After arriving in Pyongyang, Lee visited hospitals caring for children and young mothers, the Kim Dae-Jung Peace Centre in Seoul said.
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Lee had visited the North three times before, the last trip being to pay respects during the funeral of Kim Jong-Un's father, the late leader Kim Jong-Il, in December 2011.
At the time, she briefly met with Kim Jong-Un while he was receiving mourners.
The policy was however largely abandoned when a conservative administration took power in 2008 and cross-border ties soured.
A series of nuclear and missile launches by the North in recent years as well as occasional military clashes have kept tensions on a high simmer.
Seoul has stressed that Lee's visit is a personal one and she is not carrying any official message from the South Korean government.
Her entourage includes no sitting South Korean official.
Almost all talks over major issues from Pyongyang's nuclear arsenal to the resumption of official South Korean aid for the North have been shelved for years.
The visit comes just ahead of the 70th anniversary of the August 15, 1945 liberation of the pre-division Korean peninsula from Japanese colonial rule.