Zohra Hussain, 59, vice president of the women's wing of Pakistan Tehreek-e-Insaf (PTI) in the province of Sindh, was targeted by three gunmen on a motorcycle outside her home in an upmarket part of the city late Saturday.
Hussain "was leaving her home for some work when three gunmen attacked her. She thought they wanted to snatch her purse and handed it over to them but they killed her," said Firdous Shamim, a local PTI leader.
According to police official Nasir Aftab, "They shot her with one bullet near her chin and she could not survive".
The motive behind the shooting was unclear and there was no immediate claim of responsibility for the attack.
The killing came hours before voting was due to start at 43 polling stations in Karachi in a partial re-run ordered due to allegations of rigging in last weekend's polls, which marked Pakistan's first democratic transition of power.
Police said all three gunmen escaped after the attack.
Former cricket star and PTI Chairman Imran Khan was quick to pin the blame on the Muttahida Qaumi Movement (MQM) party, which represents the Urdu-speaking majority, and specifically its boss, Altaf Hussain, who lives in exile in London.
"I hold Altaf Hussain directly responsible for the murder as he had openly threatened PTI workers and leaders through public broadcasts," Khan said on his Twitter feed, describing the killing as "a targeted act of terror".
Tensions have been running high between PTI and the MQM, Karachi's dominant party, after Khan's party accused its rival of widespread vote rigging. MQM has denied the charge and announced a boycott of the re-run.
PTI and the right-wing Jamaat-e-Islami party have staged nationwide protests against the alleged irregularities.
Pakistan's election commission ordered the partial re-run due to allegations of ballot stuffing in a constituency known as NA-250 -- largely an affluent neighborhood of the financial capital.
Voting in seven constituencies in Karachi last weekend was extended by three hours after complaints that polling stations opened late in the city, which last year saw record violence linked to ethnic and political tensions.
The election saw some 50 million Pakistanis vote, with center-right former prime minister Nawaz Sharif emerging the winner nearly 14 years after he was deposed in a coup.
The Taliban, who denounce democracy, killed more than 150 people during the election campaign, including 24 on polling day.