The vital Rafah border crossing, which was re-opened early on Thursday for humanitarian cases, was later closed for security reasons.
EU and Palestinian officials said on Thursday that Israel informed European monitors that the crossing had to shut because it had a high security alert. The Israeli army had no immediate comment.
The crossing links the Gaza Strip with Egypt and has suffered numerous closures.
Officials at the terminal said that about 50 Palestinians had crossed into the Egyptian town of Rafah before the closure of the terminal and another 3,500 were still waiting.
The crossing is Gaza's only gateway to the world that bypasses Israel.
It had been closed by Israel on June 25, after a deadly cross-border raid in which Gaza armed groups killed two soldiers and seized a third, sparking a deadly offensive in the coastal strip.
Since then, the crossing has been open just once, on July 18 and 19, to allow Palestinians stranded in Egypt to cross back into the Gaza Strip.
Last November, two months after Israel withdrew from Gaza following a 38-year occupation, the Rafah border crossing began operating under a US-brokered Israeli-Palestinian agreement that allowed EU monitors.