Doctor Yaqub Khan, head of the Expanded Programme of Immunisation in Swabi district of Khyber Pakhtunkhwa province, was driving when the gunmen opened fire on his car, killing him on the spot and injuring his driver, district police official Javaid Iqbal told AFP.
Two women in the back seat were unharmed.
Another local police official Sabir Ali confirmed the incident and death.
Pakistan is one of only three countries where polio remains endemic. Attempts to eradicate it have been badly hit by militant attacks on immunisation teams that have claimed nearly 80 lives since December 2012.
The militants claim the polio vaccination drive is a front for espionage or a conspiracy to sterilise Muslims. Last year the number of polio cases recorded in Pakistan soared to 306, the highest in 14 years.
The expanded immunisation programme was launched in Pakistan in 1978 to protect children by immunising them against diseases including polio.
