Vince Hoedt, Darfur coordinator for MSF Holland, was arrested in Darfur on Tuesday.
He was flown back to Khartoum and immediately taken away by security forces.
His arrests follows the detention on Monday of the group’s country director, Paul Foreman, who has been charged with spying, publishing false reports and undermining Sudanese society.
Both aid workers were released on bail, MSF Holland said.
MSF Holland published a report in March detailing 500 cases of rape over a period of four and a half months in Sudan’s troubled Darfur region, where a rebellion has raged since early 2003.
A Sudanese man who translated for Kofi Annan during the UN chief’s visit a Darfur refugee camp on Saturday was also questioned by authorities over the rape report.
Sudan’s top UN envoy Jan Pronk condemned the arrests and said he would raise the issue with Sudanese President Omar Hassan al-Bashir.
Sudan’s attorney-general told Reuters the maximum penalty for the charges was three years in prison followed by permanent expulsion from the country.
MSF said it was outraged over the “unjustified charges” against its workers.
Geoff Prescott, the head of MSF-Holland, said the arrests “were totally unacceptable” and “seriously handicap the capacity (of MSF) to provide humanitarian assistance” in Darfur.
He accused the Khartoum government of “punishing aid workers for doing their job to help the victims of the conflict in Darfur”.
The MSF report contained anonymous accounts by rape victims, which included being held and repeatedly raped for several days, beaten and even arrested.
Pregnancy out of wedlock is illegal in Sudan, where Islamic sharia law is in force.
MSF said it could not remain silent about the atrocities in Darfur “because the scale of the violence is immense and no action is being taken to protect victims,” spokeswoman Susanne Staals said from Amsterdam.
A UN-appointed inquiry earlier this year found evidence of mass rape during the rebellion in Darfur.
The United States condemned the arrests.
“We know of the arrest of two officials of the Dutch branch of Medecins San Frontieres and we are fully opposed to this arrest and condemn it,” US State Department spokesman Richard Boucher said in Washington.
The Sudanese government has been accused of quelling the uprising with Arab militiamen known as Janjaweed, who are reported to have committed abuses including killings, rape and arson.
More than two years of conflict in Darfur has killed at least 180,000 people, many from war-induced hunger.