Khan Mohammad, the head of the Porak girls' school in Logar province, was shot dead near his home on Tuesday, said Deen Mohammad Darwish, a spokesman for the Logar governor.
"He was killed because he wanted to run the school," Darwish said.
Mateen Jafar, the education director in Logar, about an hour's drive from the capital, Kabul, said Mohammad had received several death threats from the Taliban warning him not to teach girls.
Jafar said Mohammad's son was wounded in the attack.
Education for women was banned by the Taliban government from 1996 to 2001 as un-Islamic. There are periodic attacks against schoolgirls, their teachers and school buildings.
Women have won back some rights, including education and the right to vote, since the Taliban were toppled after the US-led invasion of late 2001.
The Afghan government and its western backers have pledged to guarantee those advances, although the promise seems precarious as Afghan leaders begin a reconciliation process that includes talks with the Taliban.
Development agencies fear that western governments are focusing too heavily on plans to complete a security handover from foreign forces to Afghans by the end of 2014 without cementing gains for women, such as education.
Girls have returned to schools in recent years, particularly in Kabul, although such rights are harder to enforce in the more remote and conservative areas of Afghanistan.
Under the Taliban women were barred from access to healthcare and made to wear burqas covering them from head to toe, and only boys were allowed to attend school. Many of those customs are still widespread.
Girls have had acid thrown in their faces by hardline Islamists while walking to school and schools have been set on fire. Last year there was a spate of mysterious gas poisonings at girls' schools, including some in Kabul, in which dozens of girls fell ill.
The Taliban have not made any public comment on such attacks.
A report by aid groups in February said girls' education was at risk because of poor security, lack of funds and equipment and inadequate teacher training.
It said 2.4 million girls were enrolled at school but about 20% of those did not attend classes regularly. Those who did often faced obstacles such as open-air classrooms and journeys of up to three hours.
The report noted a shortage of female teachers – as few as one in every 100 educators in the most remote and conservative areas – which limited girls' hopes of receiving anything more than a primary education.
UK Guardian: Taliban kill head of Afghan girls' school