Mosquera said he took a legal serum during stage 15 of the race
The runner-up in the 2010 Tour of Spain has failed a drugs test.
Ezequiel Mosquera and his Xacobeo team-mate David Garcia Da Pena both tested positive for banned substance hydroxyethyl starch during the Vuelta.
Hydroxyethyl starch is a plasma substitute that increases blood volume, boosting oxygen delivery to the body.
The news comes on the day it was revealed that Tour de France champion Alberto Contador has tested positive for banned drug clenbutrerol.
The samples of Mosquera and Garcia Da Pena were tested at a World Anti-Doping Agency (Wada) accredited laboratory in Cologne.
The positive tests were revealed by the International Cycling Union (UCI) on Thursday and the Spanish pair's agent Juan Campos said they had yet to be officially informed.
Campos added that Mosquera says he took "a serum that as of now is not banned" by the UCI on stage 15 of the Tour of Spain, an individual time trial, on 15 September.
Mosquera finished second behind Italian Vincenzo Nibali in La Vuelta earlier this month.