The 24-year-old made a move from the main peloton with Dani Moreno (Movistar) on the short sharp unclassified climb just 4km from the finish, but managed to go solo with 3.5km remaining as Ben Hermans (BMC) also made it across.
Despite the peloton sitting only 20 seconds or so behind, there didn't look to be a concerted chase taking place amongst the GC teams, and the group behind Yates looked to be struggling at matching the pace the Orica rider was setting out front.
Best known for his climbing ability, Yates was able to hold off the chasers by 20 seconds and cross the line in Luintra to take a momentous victory, which comes after a difficult year that saw him banned for failing an anti-doping test.
Not only is it Yates's first Grand Tour victory, but his first at WorldTour level having joined Orica in 2014.
The day looked set to favour a breakaway or at least a reduced bunch sprint, with the summit of the route's major climb, the category two Alto Alenza, coming with 47km to go left in the stage and the uphill shortly before the finish.
But it took over 40km of racing for a large group to get away, and when it did the peloton weren't keen on letting it have much time.
The break of Andrey Zeits (Astana), Kevin Reza (FDJ), Jan Bakelants (AG2R - La Mondiale), Alberto Losada (Katusha), Gert Dockx (Lotto Soudal), Omar Fraile (Dimension Data), Mathias Frank (IAM Cycling), Valerio Conti (Lampre - Merida), José Mendes and Gregor Muhlberger (Bora-Argon18) only got 2-30 max before things began to split up.
Fraile tried his luck with a long range attack, pursued by Bakelants, Frank and Zeits, but struggled to hold on to any advantage after taking the day's KOM points and the Spaniard was caught by the three chasers with Frank then going on the attack himself with 19km to go.
That attack held out until Yates and Moreno made their move, with the Swiss rider just able to hold on and take a top-10 placing as the chasers were swamped by the peloton in final kilometre and Yates crossed the line in victory.
There were no significant changes in the general classification, with Darwin Atapuma (BMC) holding on to his lead, though Yates moved up to tenth overall with his team leader Esteban Chaves still in fifth and fellow Brits Chris Froome and Peter Kennaugh of Team Sky in third and eighth respectively.
Friday's seventh stage sees the riders face a lumpy but flowing 158.5km stage that will likely end in a reduced sprint finish.
Vuelta a España 2016, stage six Monforte de Lemos to Luintra. Ribera Sacra (163.2km)
1. Simon Yates (GBr) Orica - BikeExchange, in 4-05-00
2. Luis Leon Sanchez (Esp) Astana, at 20s
3. Fabio Felline (Ita) Trek - Segafredo, at 22
4. Ben Hermans (Bel) BMC
5. Kenny Elissonde (Fra) FDJ
6. Daniel Moreno (Esp) Movistar
7. Mathias Frank (Sui) IAM Cycling, all same time
8. Alejandro Valverde (Esp) Movistar, at 29s
9. Romain Hardy (Fra) Cofidis
10. Simon Clarke (Aus) Cannondale-Drapac, all same time
Overall classification after stage six
1. Darwin Atapuma (Col) BMC, 21-45-21
2. Alejandro Valverde (Esp) Movistar, at 28s
3. Chris Froome (GBr) Team Sky, at 32s
4. Nairo Quintana (Col) Movistar, at 38s
5. Esteban Chaves (Col) Orica-BikeExchange, st
6. Samuel Sanchez (Esp) BMC, at 1-07
7. Leopold Konig (Cze) Team Sky, at 1-12
8. Peter Kennaugh (GBr) Team Sky, at 1-14
9. Gianluca Brambilla (Ita) Etixx-Quick Step, at 1-22
10. Simon Yates (GBr) Orica - BikeExchange, at 1-28
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