Hamas shut the crossing between the Gaza Strip and Israel on Sunday after blaming the Jewish state for the assassination of one of its officials in the Palestinian enclave.
The interior ministry in Gaza, run by Islamist movement Hamas, said it was shutting the Erez crossing for an indefinite period as it investigates Friday's murder.
It did not give details on the reason behind the closure, though there was speculation authorities may be seeking to prevent those responsible for the killing from leaving.
Hamas security forces were deployed along the Gazan borders and exits by sea were also banned, officials said.
Hamas officials have blamed the Israeli intelligence agency Mossad and its "collaborators" for the killing of Mazen Faqha, 38, who was shot dead by unknown gunmen.
He was killed by four bullets from a pistol equipped with a silencer.
Israel has not commented on the shooting.
According to Israeli media, Faqha was responsible for cells of Hamas's military wing, the Ezzedine al-Qassam Brigades, in the Israeli-occupied West Bank.
On Sunday, Faqha's wife, Nahed Assida, called on "Palestinians in the West Bank and Gaza Strip to respond to this assassination."
"The price of my husband's blood is in your hands," she said during a women's conference in Gaza.
Faqha's funeral on Saturday drew thousands of Hamas supporters into the streets with chants of "revenge" and "death to Israel."
Ismail Haniya, until recently head of Hamas in Gaza, and Yahya Sinwar, who replaced him as leader, headed the procession.
According to Hamas, Faqha formed cells for the Islamist group's military wing in the West Bank cities of Tubas, also where he was born, and Jenin.
It said he played an important role in preparing two major attacks.
They included a suicide attack in the Israeli settlement neighbourhood of Gilo in east Jerusalem in 2002 that killed 19 people.
The second was a suicide bus attack later that year that killed nine people in the northern Israeli city of Safed.
They were part of a wave of suicide attacks that killed hundreds of Israelis during the second intifada, or uprising, between 2000 and 2005.
Israel sentenced him to nine life sentences plus 50 years, but he was released in 2011 along with more than 1,000 other Palestinians in exchange for Gilad Shalit, an Israeli soldier Hamas had detained for five years, and transferred to Gaza.
Israel did not immediately comment in detail on Sunday's closure but said its side of the crossing remained open.
The Erez crossing is the only one between Gaza and Israel for people. Another crossing with Israel, Kerem Shalom, is used for goods and remained open on Sunday, Palestinian officials said.
The Gaza Strip has been under an Israeli blockade for a decade. Palestinian militants in Gaza and Israel have fought three wars since 2008.
Gaza's sole crossing with Egypt has also remained largely closed in recent years.