Armed men fired at the vehicle marked with logos of the Catholic Organization for Relief and Development Aid as it travelled on a road near the town of Bentiu on Saturday and one of the workers died, the United Nations said.
"I strongly condemn the targeting of aid workers and demand that those responsible for these acts be brought to justice," Alain Noudehou, the UN's humanitarian coordinator for South Sudan said in a statement.
Nearly 100 relief workers have been killed in South Sudan, which gained independence in 2011 and spiraled into civil war two years later, Presstv reported.
Tens of thousands of people have been killed in the war initially fought between troops loyal to President Salva Kiir, an ethnic Dinka, against rebels under former vice president Riek Machar, a Nuer. Violence has spread throughout the country and now involves most of the nation's ethnic groups.
The United Nations did not say which armed force carried out Saturday's attack. Staff from another aid group, CARE International, were robbed at gunpoint while travelling in the vicinity on the same day, according to the statement.
Both rebels and the government forces stand accused of targeting humanitarian workers and sometimes deliberately blocking access to relief and hijacking food and other aid.
Noudehou called for the release of seven other aid workers detained by rebels since March 25 when they were detained in the Central Equatorial region, near the Uganda border.
The staff from a local aid group, the South Sudan Health Association, were detained as they were delivering supplies to health centers serving thousands of people, the UN said.
Lam Paul Gabriel, a spokesman for rebel group SPLA-IO, said the group had detained the workers because some of them were government spies.
"We are willing to release them (aid workers) but not all of them, those found to be (government) agents will be detained."