Bosvelt came on with Holland leading 2-1, but the Czech Republic completed a remarkable turn-around in the closing stages to win 3-2. Liverpool pair Milan Baros and Vladimir Baros scored the goals which turned the game, the most entertaining of the tournament so far.
Bosvelt, a late call-up for the injured Mark van Bommel, and his Dutch team-mates now face an early exit if they fail to get a better result than Germany in the final Group D games.
But Dutch coach Dick Advocaat has had to defend his decision of replacing Robben, who crossed for both Holland's goals.
"I substituted Arjen Robben because I wanted more grip on the midfield," he said. "(Tomas) Galasek had too much space. Now everyone is talking about my substitution that didn't work out but no one is talking about all the chances that we missed.
"Even without Robben we created four or five good chances. The substitution is my responsibility and now we have to beat Latvia and hope the Czechs do their duty against Germany."
Goals from Wilfred Bouma and Ruud van Nistelrooy put Holland 2-0 in front within 19 minutes before Jan Koller pulled one back four minutes later. The Dutch should have extended their lead before Baros hit a 71st minute equaliser.
John Heitinga's second yellow card in the 75th minute handed the initiative to the Czechs, who took the points with Smicer's winner just two minutes from time.