Initial reports said that five soldiers serving in the Spanish army were killed and three others wounded Sunday afternoon after the blast, which took place in the Marjayoun-Khiam valley.
The incident has been blamed on a car bomb or a remote controlled device.
However, one of the three injured, a 19-year-old soldier from the southwestern Spanish city of Seville, died later in hospital, AFP quoted a Spanish defense ministry spokesman as saying.
It was the first deadly attack on the United Nation`s peacekeeping force in Lebanon known as UNIFIL since the force`s mandate was expanded in the wake of a devastating 34-day war between Israel and Hezbollah in southern Lebanon.
Spain has deployed nearly 1,100 troops in southeastern Lebanon near the border as part of UNIFIL, which has 13,225 soldiers from 30 countries.
UNIFIL first deployed in Lebanon in 1978 after an Israeli invasion into the country.
A total of 250 troops and 10 civilian staff or observers working with the force have been killed since the mission was first undetaken almost 30 years ago.