Ahmad Wali Karzai, a leading power broker in the country's south, was shot dead at his home in a blow to Nato's battle against the Taliban in the area.
He was shot twice by his long-time head of security Sardar Mohammed, who was himself killed almost immediately.
The Taliban said they carried out the attack, calling it one of their top achievements in 10 years of war.
Khaled Pashtun, a Kandahar province politician, was sceptical about the Taliban claims, saying the Islamist group had claimed responsibility for many attacks in the past without much evidence of their involvement.
Sardar Mohammed's motives remain unclear, but the killing will raise questions about securing Afghanistan's top officials.
Previous assassination attempts
Kandahar police chief Abdul Razeq told reporters that Mr Mohammed had travelled to Mr Karzai's home early on Tuesday, saying he needed to show documents to his boss.
"The man carried his pistol through the security checks to Wali Karzai's room. As soon as Wali Karzai came out of the bathroom, he opened fire and shot him in the head and chest," Mr Razeq said.
The killing is the latest and most high-profile in a series of assassinations of senior politicians and security commanders across the country.
The Afghan president, who is expected to leave Kabul shortly to attend his half-brother's funeral, said the assassination reflected the suffering of all Afghan people.
"This is the way of life for the people of Afghanistan," said Mr Karzai.
"The homes of all Afghans feel this pain. Our hope is this will come to an end, and peace and happiness will come to our homes and will come to rule in our country."
Critics said Ahmad Wali Karzai was a warlord mired in corruption who was openly involved in the drugs trade and had a personal militia at his disposal.
His supporters saw him as a defender of Pashtun rights. The president repeatedly defended him, denouncing accusations that his brother was involved in criminal activities.
Security was intensified in Kandahar following Tuesday morning's shooting, as the body of Ahmad Wali Karzai was taken to a nearby hospital.
The BBC's Quentin Sommerville in Kabul says it is hard to overstate how important Ahmad Wali Karzai was in the south of Afghanistan and in Nato's efforts to combat the Taliban in their stronghold there.
Our correspondent adds that as head of the Kandahar Provincial Council, he was a staunch ally of US and allied forces in Afghanistan - to the extent that they turned a blind eye to accusations he was involved in drug-trafficking.
"[Ahmad Wali Karzai] had his unsavoury side, but he was someone we could work with and he kept a lid on things in Kandahar," a US official told the BBC.
The commander of the International Security Assistance Force (Isaf), US General David Petraeus, condemned the assassination and said Isaf would work with the Afghan government to bring anyone involved to justice.
"President Karzai is working to create a stronger, more secure Afghanistan, and for such a tragic event to happen to someone within his own family is unfathomable," said Gen Petraeus.
Ahmad Wali Karzai had survived attempts on his life before, most recently in a rocket and machine-gun attack in 2009 as his convoy was travelling towards Kabul.
The previous year, he was chairing a meeting in a government building when a bomb-filled fuel tanker exploded close by.
Although Mr Karzai escaped unhurt, six people were killed and 40 wounded in the blast.
He and other officials blamed Taliban militants for the bombing.
Books About Afghans:
Afghanistan: A Cultural and Political History (Princeton Studies in Muslim Politics)
Afghanistan: A Military History from Alexander the Great to the War against the Taliban
Afghanistan: A Short History of Its People and Politics
Ghost Wars: The Secret History of the CIA, Afghanistan, and Bin Laden, from the Soviet Invasion to September 10, 2001
Afghanistan: A Companion and Guide (Second Edition) (Odyssey Illustrated Guides)
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