Led over the line by Spaniard Jonathan Castroviejo, Movistar completed a very technical 12.6km course in a time of 14 minutes and 13 seconds - six seconds faster than both Cannondale and Orica-GreenEdge.
It was a repeat of Movistar's 2012 TTT victory in Pamplona, which also saw Castroviejo take the inaugural leader's red jersey.
Trek Factory Racing and the Omega Pharma-Quick Step team of Giro d'Italia runner up Rigoberto Uran completed the top five at nine and 11 seconds respectively.
Five seconds down to Peter Sagan's Cannondale team at the first intermediate check, Movistar rallied over the second half of the course to come home with a full quota of riders on a sunny evening in Andalusia.
On a winding course that boasted no less than 22 roundabouts, many of the main race favourites were clearly not prepared to take any unnecessary risks - especially after the whole of Fabian Cancellara's Trek team crashed on a tight, wet bend in training.
The Team Sky team of Britain's Chris Froome - himself victim of a training accident after losing control on an oil patch on Friday - conceded 27 seconds for their eleventh place while Alberto Contador's Tinkoff-Saxo squad came home for seventh place, 19 seconds in arrears.
Level on time with Tinkoff-Saxo was the Belkin outfit of Dutchmen Wilco Kelderman and Robert Gesink, two seconds ahead of Cadel Evans' BMC team.
Despite Friday's shock withdrawal of defending champion Chris Horner - pulled from the race because of dangerously low cortisol levels - the American veteran's Lampre-Merida team posted the tenth best time at 25 seconds, two seconds faster than Froome and his Sky team-mates.
Early pace-setters MTN-Qhubeka eventually shipped 29 seconds to the winners for their 12th place, but finished one second quicker than the Astana team of Fabio Aru, the Italian youngster who finished behind Quintana and Uran on the Giro d'Italia podium in May.
Spaniard Joaquim Rodriguez will start the first road stage 38 seconds down on his GC rival Quintana after Katusha toiled for 16th place on the challenging course. Another three seconds to the worse was the Garmin-Sharp team of American Andrew Talansky, Canadian Ryder Hesjedal and Ireland's Dan Martin.
FDJ, Caja Rural and Cofidis all struggled to make an impact but the only team to concede more than a minute to Movistar was the French Europcar squad, who set the 22nd and worst time some 1:03 off the pace.
With 10, six and four bonus seconds up for grabs for the top three riders in all road stages, Sunday's largely pan-flat 174.5km stage from Algeciras to San Fernando could well see a new rider seize the red jersey from Castroviejo's shoulders.
Sprinters Sagan (Cannondale) and Michael Matthews (Orica-GreenEdge) are poised at just six seconds from the race summit and the slightly uphill finale will suit both the Slovakian and the Australian.
- Sports & Recreation
- Jonathan Castroviejo
- Rigoberto Uran
- Chris Froome
- Trek Factory Racing