"A plane's primary function is not to kill. Get the difference?" MacFarlane replied to one Twitter user who suggested planes should have been banned after September 11.
When another Twitter user commented that "one person carrying a concealed handgun in that club could've ended that horror", MacFarlane replied: "Often stated, never once happened".
MacFarlane also summarised the solemn tone that took over the social media platform following the tragic news.
"Man. Twitter responses to all this on my feed are enough to send the even (sic) cheeriest soul into a deep depression," he wrote.
Fifty people were killed in the Orlando nightclub after 29-year-old Omar Saddiqui Mateen opened fire with a handgun and an "AR-15" assault rifle.
The attack also left 53 people in hospital, with several others still unidentified or missing.