Col Israel Shomer fired at Mohammed Kasbeh after the 17-year-old threw a stone at his vehicle in the West Bank.
Israel's military said at the time that Col Shomer felt his life was in danger.
But the video released by B'Tselem appears to show the teenager running away after attacking the vehicle. Soldiers then jump out and pursue him.
B'Tselem also said medical evidence suggested that he was shot in the back.
The Israeli military has not commented on the video, but the Times of Israel reported that Col Shomer had been interviewed under caution by military police investigating the shooting following its release.
The incident took place on 3 July at the Qalandiya checkpoint between Jerusalem and Ramallah, the scene of frequent clashes between Palestinian protesters and Israeli troops.
A vehicle carrying Col Shomer, commander of the army's Binyamin Brigade, was hit by stones thrown by a group of youths, shattering its windshield.
The Israel Defense Forces (IDF) said at the time that the colonel got out of the vehicle and ordered the stone-throwers to stop, firing a warning shot into the air. When the attack continued, he opened fire at the main perpetrator, it added.
The IDF stressed that Col Shomer believed he was in "imminent mortal danger" and several members of the government praised his actions.
But witnesses cast doubt on the commander's account.
Ahmed Taher, 17, who was throwing rocks beside Kasbeh, told the Washington Post after his funeral: "We were running away."
Fahdi Ziad, who works at a nearby petrol station, told the Guardian that three Israeli soldiers - two wearing a helmet, and one without one - chased after the teenagers and opened fire.
Mr Ziad said Kasbeh was about 10-15m (33-49ft) from the soldiers when he was shot. "After he fell on the ground the soldier without the helmet went and checked him and kicked him," he added.
Palestinian doctors also said the teenager was shot in the back and the side of the head.
On Monday, B'Tselem released video footage recorded by a security camera at the petrol station showing the moments before the shooting.
It shows a person believed to be Kasbeh running at the Israeli army vehicle as it passes by and throwing a stone. He then runs away as the car then brakes suddenly and at least two soldiers get out, brandishing weapons.
In a statement, B'Tselem said the footage showed Col Shomer's claim of self-defence was "unreasonable".
"There is no doubt that the shattering of the jeep's front window with a stone endangered the passengers when it happened. However, Kasbeh was shot in the back after the fact, when he was already running away and posing no 'mortal threat' to the soldiers. Feeling a sense of danger is not enough to justify any action."
The rights group also noted that the video contradicted the IDF's claim that Col Shomer had carried out "suspect-arrest procedure".
"Military open-fire regulations permit shooting at the legs of a suspect in order to facilitate his arrest. They do not permit killing him by firing three shots at his upper body," it added.
Thaer Kasbah, the dead teenager's brother, told the Associated Press that it was clear from the video that Col Shomer "wanted to kill him".
Correction 14 July 2015: An earlier headline on this story that suggested the video showed Mohammed Kasbeh being shot has been amended.
An Israeli human rights group has published a video it says contradicts the account of an Israeli army officer who shot dead a Palestinian teenager.