Despite the setback, the Azzurri still extend their lead in Group B to four points over second placed Bulgaria. The Czechs sit one point behind Bulgaria in third. All three nations have four remaining group games to play.
Italy were on the backfoot early on. Gianluigi Buffon saved a point blank header from Tomas Sivok, but an offside flag came to the visitors' rescue. Ignazio Abate made a crucial interception on Tomas Hubschman's through ball, which would have put Libor Kozak clear.
A short corner caught Italian defenders unaware as Tomas Rosicky sprinted into the box, only to send his shot into the corner flag.
Stephan El Shaarawy created Italy's first real chance on 19 minutes, pulling the ball back from the byline, but no-one could get on the end of it as it trickled across the face of goal.
The Czechs suffered an early blow when they lost David Limbersky to injury. Italy managed to restrict the hosts to long range efforts for the duration of the half. Petr Jiracek, Kozak and Michal Kadlec all had shots from 25-30 yards, but they did little to trouble Buffon.
Balotelli went for glory on the stroke of half time and ran into Czech traffic, when the better option would have been to roll the ball across to the unmarked Riccardo Montolivo.
Cesare Prandelli introduced Sebastian Giovinco for the second half, but the Italians continued to play second fiddle. Kozak was sent clear on the hour, forcing Buffon to run off his line and close the angle, before saving smartly with his feet.
Balotelli pressed the self destruct button by picking up two quick cautions to make Italy's task that much harder for the final 20 minutes.
Jiracek injured himself when he stretched to direct a late free kick off the outside of the post. In stoppage time, Italy failed to clear and the ball fell invitingly to Daniel Kolar in the box. The substitute chested the ball down and shot wide to let the Italians off the hook once again.
Czech Republic: Cech; Gebre Selassie, Sivok, Kadlec, Limbersky; (Suchy 20) Darida, (V Kadlec 76) Hubschman; Jiracek, (Kolar 86) Rosicky, Plasil; Kozak
Italy: Buffon; Abate, Barzagli, Bonucci, Chiellini; Marchisio, Pirlo, (Aquilani 77) De Rossi; Montolivo; El Shaarawy, (Giovinco 46) Balotelli
Sent off: Balotelli 72 (I)