"We will not allow them into the country," said Rajapakse, who is under international pressure to cooperate with the UN-mandated investigation.
Sri Lanka has refused to accept the authority of the UN Human Rights Council which voted in March to probe allegations that the military killed 40,000 civilians in the final months of the separatist war, which ended in 2009.
But it is the first time that Rajapakse has said UN investigators will not be allowed into the country, effectively barring them from face-to-face access to Sri Lankans wanting to testify in the probe.
Rajapakse said however that his government was cooperating with all other UN agencies.
"We are saying that we do not accept it (the probe). We are against it," he told Colombo-based foreign correspondents at his official residence.
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"But when it comes to other UN agencies, we are always ready to fully cooperate and fully engage with them," he said.
UN Secretary General Ban Ki-moon and other leaders have urged Colom