A diplomatic source that spoke on the matter said the crossing was opened following an agreement between Egypt, Israel and Gaza's Hamas rulers in mediation from Qatar in coordination with the United States.
Live footage shown on television stations showed Egyptian nurses and first-aiders examining wounded Palestinians then carrying them on stretchers to ambulances destined for the North African nation.
At least one child was visible in one of the ambulances, with officials saying around 90 of the most seriously wounded would be allowed to cross for treatment in Egyptian hospitals.
On the Palestinian side of the Rafah crossing, an AFP correspondent saw 40 ambulances enter the terminal, each one carrying two people.
The patients were to be taken to several locations, including a field hospital in Sheikh Zuweid, some 15 kilometers from Rafah. Media reports said others would be taken to a hospital in El Arish, 30 kilometers to the west with the most complex cases referred to Cairo.
After the transfer of the wounded, Egypt was to allow hundreds of foreign passport holders to cross for the first time since the Israel-Hamas war began on October 7.
Some 400 foreigners and dual nationals were expected to make the crossing on Wednesday.
Foreign governments say there are passport holders from 44 countries, as well as 28 agencies, including UN bodies, living in the Gaza Strip where 2.4 million people have endured more than three weeks of unrelenting Israeli bombardment in response to the October 7 Hamas attacks.
The tiny coastal territory has also suffered "catastrophic" shortages of food, water and electricity following an almost total Israeli blockade in response to the attacks, the deadliest in Israel's history, that killed 1,400 people, mostly civilians.
So far, Israel's retaliatory bombing campaign has killed more than 8,500 people, two-thirds of them women and children, according to the health ministry in Hamas-run Gaza.