Rosicky will also have the chance to exact revenge for Arsenal’s defeat to Barcelona in the Champions League this season.
The English side were knocked out of Europe’s premier club competition by the Spanish champions in the Round of 16, and while Rosicky was an unused substitute for the first leg of that tie at the Emirates, he started at Camp Nou.
Barcelona have six players in the current Spain squad and the 30-year-old will get a chance to put one over them.
"I can't say it's extra motivation for me," said Rosicky, according to Reuters. "It is a huge game for us, for the whole team.
"Spain are the favourites, no doubt about that, but we didn't come here to lose and we will fight."
Spain have won all three of the qualifying matches so far and top Group I with nine points, while the Czech Republic are second in the group with six.