The police said 25 men and 16 women were killed. There were around 60 passengers, including children, on the bus, mostly from the Ismaili community.
Witnesses say six men came on three motorbikes, entered the bus and fired indiscriminately for nearly 10 minutes. Most passengers were reportedly shot in the head at close range.
The bus driver is believed to have been shot first.
"There were six terrorists on three bikes. They got off the bikes, climbed the bus and opened fire using 9 mm pistols," senior police officer Najib Khan told reporters.
A pamphlet found at the site has claimed the role of the Islamic State in the killings and has threatened more attacks.
The bus reportedly belongs to the Al-Azhar Garden Colony, which is an Ismaili housing project in Karachi. It picked up people for work and dropped them off every day.
Ismaili Shiites are known for their progressive Islamic views. Their spiritual leader Prince Karim Aga Khan is a globally renowned philanthropist and business magnate.
This is the worst anti-Shiite attack since January 30, when a suicide bomber blew himself up in a mosque in the southern Shikarpur district, killing 61.
The attack sparked protests from civil society groups at some places, including the capital, Islamabad.
Anti-Shiite attacks have been increasing in recent years in Karachi and in other cities like Quetta and Gilgit. Around 1,000 Shiites have been killed in the past two years in Pakistan.
Today's attack comes as Pakistan steps up its efforts against militants following a Taliban massacre of 150 people, mainly children, in a school in Peshawar last year.
In the aftermath the government ended a six-year moratorium on executions, passed legislation to create military courts for terrorism cases, and pledged to crack down on all militant groups.
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