Israeli police said that the killing was being investigated, while the Israeli military stressed that it was not involved in the incident during which "shots were fired after stones were thrown at an Israeli car".
Route 60, which runs from Beersheba to Nazareth and connects a number of Israeli settlements, is closed to Palestinians in areas of the West Bank. It has seen a number of protests against the restrictions imposed on Palestinians by Israel.
About 500,000 settlers and about 2.5 million Palestinians live in the West Bank and areas near Jerusalem annexed by Israel after the 1967 Middle East war.
Al Jazeera's Sherine Tadros, reporting from the West Bank, said there had recently been a spike in settler violence against Palestinians.
"Many people are saying this is a direct response to what the settlers feel is a threat from the United States that there will be pressure put on the Israeli administration to stop, or at least freeze, its settlement building empire that it's constructed here in the occupied West Bank," she said.
"Many people are saying Palestinian blood has been shed as a price tag, if you like, for the setters to give that message to Prime Minister Netanyahu and his administration that he cannot touch these illegal settlements in the West Bank.
"And if he does try and dismantle them in any way or freeze construction, this will be the result."
In a report published on Thursday, the UN Office for the Co-ordination of Humanitarian Affairs in the Palestinian territories said that there had been been at least nine violent incidents involving Israeli settlers in the last week.
Israeli settlers injured five Palestinians, including three children, aged five, nine and 12, in two separate incidents of stone-throwing and physical assault, it said.
Thursday's attack in the West Bank came as Palestinians marked the 62nd anniversary of the Nakba, or Catastrophe, when an estimated 700,000 Arabs were forced to flee the creation of Israel.
Saeb Erekat, the senior Palestinian negotiator, said "the catastrophe continues" for Palestinians.
"In other conflicts, refugee rights have been honoured and respected, including the right of return, restitution and compensation. In stark contrast, however, Israel refuses to even recognise the Palestinian right of return, thus continuing to deny the refugees' basic rights.
"No state is above the law," Erekat said, calling on the international community to end Israeli "beligerence and disregard for international law".
Candle-lit vigils were planned in refugee camps on Friday evening and major demonstrations were scheduled in the Palestinian territories on Saturday, including a march to the grave of the late Palestinian leader Yasser Arafat in Ramallah.
The Palestinians recently resumed indirect peace talks with Israel, mediated by the United States, but both sides have spoken pessimistically about the chances of tangible results.