General Yusuf Issa, of the Palestinian Preventative Security force, said the crossing had been opened for only 30 minutes since its initial closure at 1pm (1100 GMT) on Friday.
"Israeli soldiers closed the crossing without informing us, without any prior notice or coordination.
"It is now closed off with cement barricades, until further notice," he said.
Effectively dividing the area into two parts, the closure has paralysed traffic between the northern and southern Gaza Strip.
Daily life has ground to a halt in the overpopulated area of 1.4 million people that is little more than 360sq km.
On Saturday, dozens of cars were backed up on both sides of the road leading to the Abo Holi and Matahen checkpoints for the second consecutive day.
Commuters have had to endure the heat and lack of drinking water as they wait for a sign that the crossing has been re-opened.
Closed to medical transports
Ambulances have been prevented from taking patients to the Shifa hospital, the largest in the Gaza Strip.
Reem Ali, a part-time student at al-Quds Open University in Khan Yunus, is stranded in Gaza city because of the closure.
"My mother is sick and needs me to take her to the hospital. Her medical insurance papers are with me. It's a catastrophe."
She says the closure forced her to miss a final exam for her master's degree.
A humiliating move?
Abu Ali al-Farra, a Palestinian police officer, said the soldiers manning the checkpoint, who control it remotely from pillboxes fortified by cement blocks, electric fences and sandbags, have been taunting Palestinian commuters waiting for hours to get through.
"The soldier yells out through his megaphone for us to get back in our cars so he let us through. He says if we don't get inside our cars in exactly two minutes he'll close the checkpoint forever. So we do, and once we're in, the checkpoint stays closed anyway," he said.
Rights groups say freedom of
movement has not improved
Al-Farra has been waiting seven hours since early Saturday morning to return to his home in Khan Yunus from his night shift in Gaza City.
"He's playing with our nerves, trying to humiliate and punish us. He lets through a few trucks of commercial goods being sold to the Israelis but lets the people suffer."
The closure comes despite promises by the Israeli government during February's Sharm al-Shaikh summit to ease travel restrictions in the Gaza Strip.
According to a report published by the Israeli human rights group B'tselem last month, there has been almost no improvement in the movement of Palestinians to and from Gaza, or in the movement of goods, since the summit.
Palestinians usually wait at
crossings for several days
The group says the closures are a form of collective punishment, illegal under international law.
"Almost all the restrictions on movement are imposed on entire categories of people, based on sweeping criteria, without checking if the individual poses a security risk, and without weighing the harm the person will suffer, or if less harmful alternatives are available," said the group.
An Israeli army spokesperson could not be reached for comment on Saturday. In the past, such closures have been called security measures.
On Saturday, Palestinian Interior Minister Nasser Youssef said resistance fighters had agreed to halt a spate of mortar and rocket attacks on Jewish settlements in the Gaza Strip.
A recent flare-up of violence has threatened a three-month ceasefire.
On Friday, Palestinian resistance groups Hamas and the al-Aqsa Martyrs Brigades carried out an attack on an illegal Jewish settlement in Gaza in which a fighter was shot dead by Israeli occupation forces.
A Hamas spokesman said the attack was in response to Israeli aggression.
The clash came one day after Israel's defence chief Shaul Mofaz ordered the army to use all necessary means to stop home-made mortar and rocket launches on settlements.
Since the signing of the Sharm al-Shaikh ceasefire agreement on 8 February, 23 Palestinians have been shot and killed by Israeli soldiers - 11 of them children, according to the Palestinian Red Crescent.
Five Israelis have also been killed in the same period.