Two of Nakamura's bodyguards, the driver of the auto, and another person were killed on the spot, said Attaullah Khogyani, spokesman for the Nangarhar Province's governor.
He underwent surgery at a local hospital but died of shortly after while being airlifted to the Bagram airfield hospital in the capital, Kabul, for further treatment, said Gulzada Sanger, the hospital spokesman.
Tetsu Nakamura, 73, was the head of Peace Japan Medical Services - known as Peshawar Kai in Japanese - and had been working in the region since the 1980s, when he began treating patients with leprosy in Peshawar in neighbouring Pakistan.
Afghan security force personnel inspect an area around a vehicle, following an attack in Jalalabad on Wednesday.
Nakamura was seriously wounded and was reported to be in critical condition immediately after the attack in the provincial capital.
Officials told RFE/RL on December 4 that gunmen opened fire at a auto carrying Tetsu Nakamura, a 73-year-old physician who headed a Japanese charity focused on improving irrigation and agriculture in Afghanistan, in the provincial capital, Jalalabad.
He came to Afghanistan after a Japanese colleague, Kazuya Ito, was abducted and killed.
Nakamura also described a variety of his organisation's projects to help Afghans, including the construction of wells and irrigation canals, as well as health services.
A spokesperson for Afghan President Ashraf Ghani called Nakamura "one of the closest friends of Afghanistan".
No one immediately claimed responsibility for the attack, the second in as many weeks targeting aid workers in Afghanistan.
Hundreds of Afghans posted photographs of Nakamura on their social media pages, condemning the killing and underscoring how respected the Japanese physician was.
The attack comes as humanitarian groups remain on high alert just days after an American aid worker for the UN Development Program was killed when his vehicle was targeted in a bombing in Kabul on November 24.
In Fukuoka, southwestern Japan, where the charity is based, the organization's spokesman Mitsuji Fukumoto lauded Nakamura's work.
The Taliban, who along with the Islamic State group, operate across the province, denied involvement in Wednesday's attack.
The gunmen fled the scene and police have launched a search operation to arrest them, Sohrab Qaderi, a member of the governing council in the province of Nangarhar told Reuters, adding he believed Nakamura had been targeted for his work.
Aid agencies and non-governmental groups have been targeted in Afghanistan's almost 20-years-long war.
The Taliban control or hold sway over almost half of Afghanistan, staging near-daily attacks that target Afghan forces and government officials but also kill scores of civilians.
He "dedicated his life to helping and cooperating with our people", spokesperson Sediq Seddiqi said.