The gunmen fired at polio workers as they administered polio drops to children in the Kamangara area of Nawagai tehsil near the Pak-Afghan border, said Assistant Political Agent (APA) Nawagai, Fayaz Ali Sherpao.
One polio worker died on the spot while another was injured, Sherpao added. The injured worker was rushed to a nearby hospital for treatment.
The Tehreek-i-Taliban Pakistan (TTP) took responsibility of the killing.
The polio team in Mansehra was vaccinating children on the second day of a three-day anti-polio drive in the district when they were attacked.
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Pakistan is one of only three countries where the crippling disease remains endemic.
Islamist militants consider anti-polio vaccines as a Western conspiracy to sterilise Muslims. Scores of health workers in Pakistan have been killed in the last two years as a result.
Last year, the number of polio cases recorded in Pakistan soared to 306, the highest in 14 years.