The Rafah crossing, in what was once a city of a quarter of a million people that Israel has since completely demolished, is the only route in or out for nearly all of the Gaza Strip's more than two million residents.
It was largely shut for most of the war, and reopening it to allow access to the outside world is one of the last significant steps required under the initial phase of a US-brokered ceasefire reached in October.
An Israeli security official said Rafah had opened about 9am "for both entry and exit".
A Palestinian source said that on the first day 50 Palestinians were expected to re-enter the coastal Gaza Strip.
Egyptian and Palestinian sources said the 50 Palestinians returning to the enclave were being processed at the Palestinian Israeli-controlled side of the border but it was unclear when they would enter the Gaza Strip, pending Israeli security checks.
Five patients seeking to leave the strip for medical treatment, each escorted by two relatives, were driven to the crossing compound from the Gaza Strip side in a vehicle escorted by World Health Organisation personnel, health officials said.
Later on Monday, Palestinian and Egyptian sources said Gazan patients had crossed into the Egyptian side of the passage and would be directed towards Egyptian hospitals.
Palestinian officials blamed delays on Israeli security checks.
Israel's military had no immediate comment.
"The crossing is a lifeline for Gaza, it is the lifeline for us, the patients," said Moustafa Abdel Hadi, 32, who receives kidney dialysis at al-Aqsa Martyrs Hospital and is one of 20,000 Gazans hoping to leave for treatment abroad.
"We want to be treated in order to return to live our normal life."
Israel seized the border crossing in May 2024, about seven months into the Gaza Strip war.
Since then, it has largely been closed apart from a brief period during an earlier truce in early 2025.
Reopening the crossing was one of the requirements under the October ceasefire that outlined the first phase of US President Donald Trump's plan to stop fighting between Israel and Palestinian Hamas militants.
In January, Trump declared the start of the second phase, meant to have the sides negotiate the shattered enclave's future governance and reconstruction.
Even as the crossing reopened, Israeli strikes killed at least four Palestinians on Monday, including a three-year-old boy, in separate incidents in the north and south of the strip.
The Israeli military had no immediate comment on the incidents.
In the war's early months before Israel shut the crossing, about 100,000 Palestinians exited to Egypt through Rafah.
Although Egypt has repeatedly made clear it will not allow a large-scale exodus, the route is seen as vital for wounded and sick Palestinians to seek medical care.
While it was closed, only a few thousand were allowed out for medical treatment in third countries through Israel.
Palestinians seeking to cross at Rafah will require Israeli security approval, three Egyptian sources said.
Reinforced concrete walls, topped with barbed wire, have been installed along the crossing area, the sources said.
At the crossing they will have to pass through three separate gates including one administered by the internationally recognised Palestinian Authority under supervision of a European Union task force but controlled remotely by Israel.
