Israeli tanks dramatically rolled into the main entry point for aid entering via Egypt and took “operational control”.
Aid agencies said its closure, along with the other main crossing Kerem Shalom, risked a disaster for millions.
UNICEF has said Gaza is on the “precipice of famine”.
UN officials accused Israel of not allowing humanitarian workers access to the Rafah crossing.
Kerem Shalom was closed after a Hamas rocket attack last weekend killed four Israeli soldiers. UNICEF’s Tess Ingram said: “Nothing has got into Gaza for the last 24 hours, which is devastating.
“No aid coming in means that in a day or two, distribution of that aid will stop and we’ll run out of supplies that remain in Gaza.
“I was at that crossing just a few weeks ago and it was a hubbub of activity with people coming in and out but also an enormous amount of aid moving in. It’s heartbreaking to see it empty and with tanks there.”
UN Palestinian refugee aid agency UNRWA called the situation “extremely concerning”.
Jens Laerke, from the UN’s humanitarian office, said it had just a day of fuel left to sustain its entire Gaza operation.
He said: “Rafah is in the crosshairs. IDF is ignoring all warnings about what this could mean for civilians. I think it’s fair to say that the reports that we get from colleagues on the ground is that panic and despair has taken hold: People are terrified.”
Israel said that both crossings would remain closed “for the time being”.
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