Witsel, who joined the Russian champions for 40 million euros from Benfica just before the September 3 transfer deadline, has pulled a hamstring in training, a Zenit spokesman said on Monday.
Italy left-back Domenico Criscito, Russia midfielder Sergei Semak (pictured, right, with Witsel) and Portugal international Danny will also miss the Group C opener in Spain.
Zenit's other high-profile acquisition, Brazil striker Hulk, is fit and should make his Champions League debut for the Russian club.
AC Milan host Belgian champions Anderlecht in the other Group C match on Tuesday.
- Domenico Criscito
- Sergei Semak