State Police allege that Justin Holmes, 21, of Clinton was riding with Jessica Holes, 24, also of Clinton, when he stabbed her from the back seat in the back and neck.
There were two other occupants in the vehicle.
One passenger, Kovarus Hills, 24, of Clinton was stabbed in the hand as he tried to stop the attack, police said. The other occupant was an infant who suffered minor injuries in the crash.
No information was available as to what provoked the alleged attack.
The brother is charged with second-degree murder and aggravated second-degree battery.