Yushchenko fell gravely ill during the 2004 presidential campaign while he was a leading opposition candidate. He was later diagnosed with a massive dioxin poisoning, which disfigured his face.
The probe has been under way since 2004, but no charges have been filed.
Yushchenko has claimed he knows who organized the crime, though he refuses to provide more detailed information.
"We should wait until the investigation is over," he told reporters before being questioned. "I am convinced that, based on the materials the prosecutor's office possesses, we will hear a lot of new reasons that will surprise citizens."
Yushchenko already testified in the case in 2005, and it was unclear why he was summoned again. Yushchenko told reporters that he was happy that prosecutors were now handling the investigation with renewed vigor and expressed hope the case would soon be solved.
The Prosecutor General's Office declined to comment, as the investigation was ongoing. A spokesman for prosecutors said Yushchenko was likely to be questioned by Halyna Klymovych, the senior investigator who is heading the case.
Many Ukrainians point the finger at Russia since Yushchenko was running against a Kremlin-backed candidate in the election and Russia is reportedly one of the few countries that produces dioxin of the formula with which he was poisoned.
Yushchenko has accused Moscow of stalling the probe by refusing to extradite key figures and provide samples of Russian-made dioxin for tests.
The president also suggested that some figures involved in the case have betrayed national interests.
"There will be very unpleasant things for specific people who, it seemed, were defending Ukraine's national interests, or at least were supposed to defend them," Yushchenko said.
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