The five-year-old, named Australia's horse of the year last season, started the $1.26 favourite and delivered a commanding performance under jockey James McDonald. She held off a brief challenge from Ceolwulf before sprinting clear.
Trainer Chris Waller described the win as straightforward, coming after what he called "an interesting week". Waller had adjusted Autumn Glow's training in anticipation of front-running rival Pride Of Jenni, who was scratched on Thursday with a leg infection.
"I was watching Ceolwulf and I was watching it with Darren [Beadman] and I said, 'Oh, are we in trouble?' And he said, 'No'," Waller recounted.
Waller praised the mare's professionalism, saying: "Her work's been fantastic and she's just an absolute professional."
The victory takes Autumn Glow to 14 wins in 15 career starts, cementing her status as one of Australia's most successful racehorses. The King Charles III Stakes is run over 1609 metres.