Investigators have determined the Friday shooting was accidental and have not charged the 16-year-old, WFTV-TV, Orlando, reported Saturday.
Police said the two boys were home alone when the older boy suspected an intruder was in the home.
"He heard some noises and he called out for his brother and he didn't answer and so then his brother startled him," said Orlando Police Department Detective Mike Moreschi.
When the older boy realized his mistake, he called 911. The younger boy was taken to Arnold Palmer Hospital where he died.
"He's devastated, obviously. His parents are devastated by what happened and it's just a really bad situation for the family," Moreschi said.
Investigators said they are evaluating the case for signs of possible negligence. It was unclear where in the house the gun was located and whether it was locked up, WFTV reported.