Young Sumit Nagal, who was named as a reserve in the side, will now be making his Davis Cup debut and will be a playing member of the squad.
"Rohan Bopanna has informed the Chairman Selection Committee that due to a knee injury which he suffered during US Open, he has been advised two weeks rest from competition. Hence he has requested for withdrawal from the Davis Cup tie against Spain which is going to be held in Delhi from 16-18 September 2016. His request was considered and accepted by the Committee," an AITA release said.
"It was also decided by the Committee to include Sumit Nagal in the four playing member team for the Davis Cup tie against Spain," the release added.
Bopanna was knocked out in the second round of the men's doubles while he made a quarterfinal exit from the mixed doubles competition at the US Open, the last Grand Slam of the season.
India are already without their top players such as Yuki Bhambri and Somdev Devvarman, who are recuperating from their respective injuries.
India's Davis Cup coach Zeeshan Ali said Bopanna's injury was "unfortunate".
"That is part and parcel of professional. Unfortunate it has happened one week before the tie," he told PTI from Bangalore.
Asked if Saketh will now play doubles with Leander Paes, Zeeshan said the combination has to be worked out.
"We don't know as of now. We will have to wait and see. We can't make Saketh sit down. How can we make him sit. He is our best singles player and he recently played in the US Open main draw as well after qualifying. We have to see," he said.
On the contrary, Spain are coming full strength, led by 14-time Grand Slam champion Rafael Nadal. They did not need even their best players to beat India but are are still attaching so much importance to the competition.