Serbia, which opposes Kosovo's independence, has warned the takeover of the border crossings could spark violent clashes.
"Let me be very clear there can be no turning back. NATO has spent 12 years ensuring stability and security and we will not allow that achievement to be put at risk," Rasmussen told reporters in Pristina on Thursday.
"KFOR troops have to act in self-defence and...they also have to act if it is necessary to maintain a safe and secure environment," he said, referring to NATO's 6000-strong peacekeeping force in Kosovo.
In July Pristina tried to install police and customs at the two crossings on its border with Serbia, but armed Serbs drove them back, burning down one border gate and leaving NATO soldiers scrambling to intervene. One ethnic Albanian policeman was killed during the riots.
This time, Rasmussen said his troops would be prepared and urged both sides to show restraint.
"Unilateral moves are unhelpful so I look to all the communities and all the leaders to show utmost care in their actions in the coming days," Rasmussen said.
Kosovo wants to send its police and customs officials on Friday to the largely lawless northern territory which has a sizeable Serb community, to assert its authority three years after its Albanian majority declared independence from Serbia.
The European Union police and justice mission, EULEX, will also deploy its own police and customs officers to the border crossings.
Local Serbs set up roadblocks on the main roads in the area on Thursday, including a barricade on the main bridge in the divided city of Mitrovica, where the Ibar River divides the Albanian and Serb districts.
Serbian President Boris Tadic said the deployment of Kosovo's customs and police had not been agreed at European Union-sponsored talks between Belgrade and Pristina, which started in Brussels in March.
"I must express my great concern over (Pristina's) unilateral moves... because they are seriously endangering the peace and security of the whole region," Tadic told a forum in Belgrade on Thursday. "This must be prevented."
Meanwhile, Kosovo accused Serbia of planning to use violence to sabotage Pristina's plan to take over the border posts.
"In the north there is still no rule and law. We will not surrender to illegal and criminal structures," Kosovo Prime Minister Hashim Thaci said after meeting Rasmussen.
UN Secretary-General Ban Ki-moon urged Kosovo and Serbia to avoid actions that could exacerbate tensions in northern Kosovo.
"I call on all concerned to refrain from unilateral actions which could escalate tensions in the area," Ban told a news conference.
In a move likely to stoke tensions further, the Serbian Orthodox Patriarch Irinej and top bishops sought to rally Kosovo Serbs on Thursday.
"We have lived to be deprived from what is most sacred from us. There's no land that's so sacred to Serbia like Kosovo," Irinej said in his sermon in a church in northern Mitrovica.
The Patriarch urged Kosovo Serbs to use non-violent means of protest.
Serbia cherishes Kosovo as its historic heartland and most of its medieval monasteries and churches are located there. The conservative Serbian Orthodox Church is vehemently opposed to Kosovo's independence.
Serbia is under pressure from the European Union to mend ties with Kosovo in order to gain coveted EU candidate status, a step to eventual accession. But the government in Belgrade also has one eye on a parliamentary election due next year and Kosovo is high on its agenda.
Serbia lost control over Kosovo in 1999, when NATO bombed to halt Serb atrocities and ethnic cleansing in a counter-insurgency war under then Yugoslav strongman Slobodan Milosevic.
Since 2008, the new state of around 1.7 million mostly ethnic Albanians has been recognised by more than 80 countries, including the United States and most of the EU.
