The attack came as witnesses said most residents in the southeastern province responded to calls for a general strike on Monday.
Hadramawt has been shaken since December 20 by protests against Yemen's central government after the army killed tribal chief Said Ben Habrish and his bodyguards at a checkpoint.
The simmering tension erupted again on Sunday when a tribesman was killed at an army checkpoint.
"Gunmen overnight blew up the pipeline linking Masila oilfield to Al-Daba port" in the town of Shahr on the Gulf of Aden, a security official told AFP.
Witnesses reported seeing flames erupting from the site of the attack, and an oil industry official said the flow of crude along the pipeline had come to a halt.
Ahmad Bamaezz, a tribal chief in the area, said youths from a tribe in Hadramawt were behind the attack.
The same pipeline had come under attack on December 28. It usually pumps about 120,000 barrels per day.
The latest attack comes amid rising tensions between Yemeni authorities and southern secessionists, allied with a group of tribes from Hadramawt, an Al-Qaeda stronghold.
Attacks on oil and gas pipelines in Yemen are frequent, and Oil Minister Ahmad Dares said last month that sabotage had cost the country $4.75 billion (3.5 billion euros) between March 2011 and March 2013.
NJF/NJF