While the Australian squad set the fastest time after crossing the finish line with three teams left to finish, ultimately it was Trek-Segafredo who took the victory and Boels-Dolmans narrowly claiming second.
Mitchelton-Scott made a rapid start to the team time trial - as the squad negotiated the city streets before hitting the back roads.
Moniek Tenniglo was the first rider to swing off, before Australian Lucy Kennedy gave one last pull on the front, leaving countrywoman Spratt, along with Annemiek van Vleuten, Grace Brown and Sarah Roy to race across the line.
"We’re really happy and proud with that result," Spratt said.
"We didn’t put a lot of emphasis on the team time trial, we thought we’d have a solid performance, but we were withing 10 seconds of the win.
"So, I think we’re all super, super proud, every single person contributed to this result, which makes me really proud and it shows that everyone on the team is in great shape. It’s a super start to the week ahead."
Mitchelton-Scott Sports Director, Alejandro Gonzalez-Tablas was also very proud of his team.
"We have done way more than what we were aiming for and we can be pretty happy with that result. We wanted to be there in the mix, we were maybe expecting to be 20 seconds down at the finish," Gonzalez-Tablas said.
"But to come home only five seconds behind the winner, we can be very proud of that ride and it shows we are in a good place for the stages coming up."
Giro Rosa Stage 1 (TTT) - results:
1. Trek-Segafredo 20:05:99
2. Boels-Dolmans 20:08:75
3. Mitchelton-Scott 20:10:45