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Anacona broke clear on the 8km, first category climb to the finish of the 185km stage from Carboneras de Guadazon to Aramon Valdelinares to move into fourth place overall with Kazakstan's Alexey Lutsenko and Italian Damiano Cunego finishing second and third respectively.
Quintana reacted to an attack from Alberto Contador with a kilometre left to take the overall lead from Movistar teammate Alejandro Valverde.
However, it was a disappointing finish for Chris Froome and Valverde as they lost 23 seconds on the final climb.
Quintana now leads Contador by three seconds with Valverde eight seconds back. Anacona is just one second further back, but having gained time on his rivals in recent days, Froome dropped to 28sec behind.
"This is a very important moment in my career. It is my first stage victory," said Anacona.
"It is a shame that I couldn't take the overall lead as well, but I just struggled a little on the final stretch but that is a motivation to keep working going forward."
The conditions changed drastically in the first full day away from the searing heat of the southern region of Andalusia as torrential rain fell throughout the stage.
Anacona was one of 31 riders to make an early breakaway from the peloton and he surged away from Spaniard Dani Moreno on the final climb to ease to victory.
Behind him a tactical battle began amongst the race favourites as Contador attacked in a bid for the leader's red jersey.
Quintana and Joaquim Rodriguez reacted in time to catch the two-time Vuelta winner, but Froome and Valverde were left behind.
"The team worked very hard today and my legs felt good. The conditions favoured us to allow me to become the leader," said Quintana.
"I didn't do a lot. I finished along with the other favourites and we continue almost like it is 0-0.
"The idea is to stay in red all the way to Santiago, but if not then hopefully I will be after the last day.
"The objective is this year to make the podium at least to complete the podium at all three grand tours at 24 years old and go for the Tour de France another year."
There is a brief respite for all the riders in the first rest day of the race on Monday before the action gets back underway with a 36.7km individual time trial from Real Monasterio de Santa Maria de Veruela to Borja on Tuesday.
Tour of Spain results and standings after Sunday's ninth stage, a 185km ride from Carboneras de Guadazaon to Aramon Valdelinares on Sunday:
Stage
1. Winner Anacona (COL/LAM) 4hr 34min 14sec
2. Alexey Lutsenko (KAZ/AST) at 45sec
3. Damiano Cunego (ITA/LAM) 50
4. Javier Moreno (ESP/MOV) 1min 04sec
5. Peio Bilbao (ESP/CJR) 1:12
6. Jerome Coppel (FRA/COF) 1:21
7. Ryder Hesjedal (CAN/GRM) 1:33
8. Adam Hansen (AUS/LTB) 1:45
9. Bob Jungels (LUX/TRE) 1:49
10. Fabio Felline (ITA/TRE) 2:08
Overall
1. Nairo Quintana (COL/MOV) 35hr 58min 05sec
2. Alberto Contador (ESP/TIN) at 3sec
3. Alejandro Valverde (ESP/MOV) 8
4. Winner Anacona (COL/LAM) 9
5. Chris Froome (GBR/SKY) 28
6. Joaquin Rodriguez (ESP/KAT) 30
7. Fabio Aru (ITA/AST) 1:06
8. Robert Gesink (NED/BKN) 1:19
9. Rigoberto Uran (COL/OPQ) 1:26
10. Warren Barguil (FRA/GIA) s.t.
