Brett McGurk, the senior US envoy to the international coalition fighting the Islamic State group, said Idlib province had become "the largest Al-Qaeda safe haven".
In comments to the Washington-based think tank the Middle East Institute last week, McGurk criticised some US partners for sending in "tens of thousands of tons of weapons" to Syria. He did not name which partners.
He added that the way those foreign fighters come in to Syria "may not have been the best approach" because Al-Qaeda had taken full advantage of it.
But Turkish presidential spokesman Ibrahim Kalin said it was not Turkey which controlled Idlib province.
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"Attempting to associate Turkey with that terror organisation in Idlib, making such an implication is unacceptable," Kalin told the TvNet broadcaster.
"Why? Because we are not controlling Idlib."
Idlib is the only province in Syria that remains entirely beyond regime control after having been captured in 2015 by an alliance of jihadists and rebels.
Foreign ministry spokesman Huseyin Muftuoglu added that Ankara had protested against the "provocative" comments, without elaborating.
McGurk has been a frequent target of the ire of the Turkish government for comments supporting Kurdish groups labelled as terror organisations by Ankara.
Turkish Foreign Minister Mevlut Cavusoglu has called for McGurk to be fired, claiming he was backing Kurdish militants in Syria.
In June, McGurk had talks with Turkish officials in Ankara.
The alliance that captured the Idlib province was dominated by Al-Qaeda affiliate Al-Nusra Front and the rebel Ahrar al-Sham, a key Islamist faction backed by neighbouring Turkey and Gulf states.
Turkey's NATO allies in the early years of the civil war accused Ankara of turning a blind eye -- or even aiding -- the rise of Islamist groups in Syria including Islamic State jihadists. But Turkey always angrily rejected the charges.