"North Korea's action creates a barrier to the stable operation" of the complex, the South Korean Unification Ministry said in a statement, urging its neighbor to "immediately normalize" the entry and exit process.
And South Korean Defense Minister Kim Kwan-jin said military action could be taken if the safety of the South Koreans in the zone were to come under threat.
"If there is a serious situation, we are fully ready, including military measures," he said at a meeting of lawmakers, the semiofficial South Korean news agency Yonhap reported.
The North has blocked the crossing into Kaesong before. In March 2009, also during joint U.S.-South Korean military drills that it said were a threat, Pyongyang shut the border, temporarily trapping hundreds of South Korean workers in the industrial complex.
It allowed many of the stranded workers to return to South Korea the next day, and fully reopened the border about a week later without explaining its reversal. It was unclear whether the latest drama over Kaesong would play out in similar fashion.
At the start of the day, when the North informed the South that it would prevent new entries to the complex, there were 861 South Korean workers in there, according to the Unification Ministry. The North said it would continue to let people leave the zone.
Hundreds of workers rotate in and out of Kaesong each day in a series of scheduled entries and exits. Many of them stay there for several nights.
During the late morning and early afternoon exit windows, only a trickle of workers was seen returning to South Korea from Kaesong, far fewer than the scores who were registered to leave at those times. South Korean authorities didn't immediately provide an explanation for the discrepancy, saying the individual companies decide when to send workers back.
Kim Kyong-sin, the manager of a textile manufacturing company in Kaesong who came back into South Korea on Wednesday, said some people were staying in the complex because "they are worried they might not be able to come back in."
During the March 2009 crisis, many South Korean companies with operations in the zone chose to keep more workers there to compensate for those not being allowed in. Kim said he was scheduled to go back into Kaesong on Thursday, but wasn't optimistic.
"I think if this continues there, business will be affected," Kim said. "I think the damage will be serious."
Kerry calls North 'reckless'
U.S. and South Korean officials have kept up their criticism of the North's actions in recent days.
John Kerry, the U.S. secretary of state, warned Tuesday that the United States will not accept North Korea as a "nuclear state."
"The bottom line is simply that what Kim Jong Un is choosing to do is provocative. It is dangerous, reckless," Kerry said during a joint briefing in Washington with South Korean Foreign Minister Yun Byung-se.
"And I reiterate again the United States will do what is necessary to defend ourselves and defend our allies, Korea and Japan," Kerry added. "We are fully prepared and capable of doing so, and I think the DPRK understands that."
The North has conducted three nuclear bomb tests, the most recent in February. It has said that its nuclear weapons are a deterrent that are no longer up for negotiation.
Carlin said North Korea's longer-range missiles may not be ready to be used for three to four years, and its nuclear program is a "low-level threat" at this point. He said Washington's most recent moves could be caused by it seeing some sort of movement around North Korean missile facilities, or it could be "misreading and over-reading North Korean propaganda but fulfilling their obligation to be on guard and prepared."
"We're going to get out of this particular crisis, it seems to me, without anything really blowing up," Carlin said. "But down the road, things are going to get more serious."
"What we should be looking at, really, is the decisions and the policies and the approach that we're going to have to take over the next four or five years to deal with these things," he added. "Because for the last five years, we really didn't do a very good job of doing that."