Police spokesman Dominant Kawcgwok said a group of mostly women and children -- fleeing violence further north -- were travelling in a lorry between the capital Juba and the southern city of Yei when it "fell into ambush".
"We lost something like 21 people," and another 20 were wounded, he said.
The government has blamed the attack on rebel forces supporting former vice president Riek Machar, who fled to Khartoum after fighting broke out in Juba in July.
UNMISS "is extremely concerned by the continuing deterioration of the security situation in Yei, Central Equatoria, where the Mission continues to be denied access," read the statement.
In another incident on Monday, three passenger buses travelling between Juba and the Ugandan capital Kampala were attacked by unknown gunmen, Kawcgwok said. He said the attackers torched one of the buses, but that none of the passengers were harmed.
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