MSF says no reasons were given for the order to stop work in Maradi region, but local media say it is suspected of having links with Tuareg rebels.
The charity said it was continuing to offer a reduced level of help while talks continued with the government.
The group provides treatment and food aid for ten of thousands of children in Niger, where malnutrition is rife.
In 2005, it suffered a major food crisis which aid organisations said the government was trying to hide.
Niger's Radio Anfani said MSF was suspected of helping a group fighting a rebellion in the north of the country, the Niger Movement for Justice (MNJ).
The MNJ is made up of nomadic people from the Tuareg ethnic group, which has long complained of being marginalised by the government.
Since last year, a series of attacks by the group on government facilities has since claimed the lives of dozens of soldiers.
The rebels recently called for 30% of the revenue from the lucrative uranium mines to be spent in their region, and have threatened to disrupt mining operations unless their demands are met.
The government has dismissed the MNJ as drug smugglers, arms traffickers and bandits with no political agenda.
MSF withdrew from the north of Niger for several months in 2007 after three of its vehicles were hijacked by rebels near Agadez.