“I was really looking forward to this match as it is one that will decide our fate in the tournament,” Khune said on Saturday.
“I am very sad and disappointed that I will not be playing. It is not nice missing such an important fixture for your country, but I believe Moeneeb Josephs and Siyabonga Mpontshane are capable of doing a great job.
“These things happen – injuries are part and parcel of the game and hopefully I will be back in the side for the following match.”
Khune suffered an ankle injury on Wednesday night and was taken for a scan the next day.
He trained on the side-lines at Bafana Bafana’s final training session at the Clyde Pinelands grounds but was ruled out of Sunday’s match at the Cape Town Stadium.
“Hopefully we still have more matches after this one and Khune will be able to join us,” coach Gordon Igesund said.
“For now his health is important. Rather not use him now and have him available for the latter stages of the tournament instead of playing him tomorrow and losing him for a longer period of time. The risk is too high.”
Khune said he was confident of a win against the Super Eagles.
“We stand a very good chance of getting victory over Nigeria. We just need to go out there and apply ourselves like we have been at training and in the past matches,” he said.
“I have been receiving messages of support for the team from all over the country, fans saying they will be at the stadium and that is what we need, it is a good morale booster to know the country is behind us.
“With that kind of support and the belief that the players have, we should be able to emerge victorious after 90 minutes and go on to the next round, but it won’t be easy because Nigeria is a good side too. We are confident though that we will make it.”
Josephs has been capped 23 times for Bafana Bafana and last played for the national team in a friendly match against Mozambique in September 2012.
In the other Group A match, Mali face Mozambique at the Athlone Stadium in Cape Town. The match also kicks off at 7pm.