The Omega Pharma-QuickStep team - which comprises Tony Martin, Sylvain Chavanel, Michal Kwiatkowski, Niki Terpstra, Kristof van de Walle and Peter Velits - won the men's team time-trial in one hour four minutes 16.81 seconds, just 0.81 seconds ahead of Orica-GreenEdge after 56.8 kilometres of racing.
Team Sky, which included Tour de France champion Chris Froome and fellow British rider Geraint Thomas, finished in third place, 22.55sec behind the leaders.
The team time-trial events are for trade teams, with riders having reverted to national colours for the World Championships in Florence, which conclude on Sunday.
Germany's Martin will now turn his attention to Wednesday's individual time-trial, in which he will hope to defend his title against British Olympic champion Sir Bradley Wiggins, Switzerland's Fabian Cancellara and Taylor Phinney of the United States.
The other team to defend its time-trial title was Specialized-Lululemon, with a women's squad which boasts British rider Katie Colclough. The six-woman team triumphed in 51mins 10.69sec, with the Rabo squad second, a full 1:11.09 off the pace. The British Wiggle Honda squad - which is backed by Wiggins and includes Olympic team pursuit champions Dani King and Joanna Rowsell - was a further 2:33 behind in sixth.