The Syrian province of Idlib and surrounding areas are the last major enclave held by rebels opposed to Syrian President Bashar al-Assad, a close Iranian and Russian ally.
Assad is reportedly preparing a phased offensive to regain the province.
The US President wrote on Twitter: “President Bashar al-Assad of Syria must not recklessly attack Idlib Province.
“The Russians and Iranians would be making a grave humanitarian mistake to take part in this potential human tragedy.
“Hundreds of thousands of people could be killed.
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“Don’t let that happen!”
The message is being seen as a red line, with suggestions the President may retaliate to any attack on the province.
Trump’s tweet comes a few days after Russia declared part of the Mediterranean would be “dangerous” while they hold a massive naval exercise.
Russian sources have claimed the military drills are needed ahead of the final assault on Syria’s rebels in Idlib.
Kremlin spokesman Dmitry Peskov said: “This hotbed of terrorists in Idlib does really not bode anything good if such inaction continues.
“The situation in Syria has a significant potential to become more complicated and the situation around Idlib leaves a lot to be desired.”
Syrian Foreign Minister Walid al-Moualem said last week: “We will go all the way in Idlib.”
Russian Deputy Foreign Minister Mikhail Bogdanov denied the upcoming naval drills were in relation to the situation in Syria’s Idlib and had been planned in advance.
But the exercise appeared aimed as a warning to the West and UN forces against hitting Syrian government positions with tactical strikes.
In April, the US, UK and France launched over 100 missiles which they say targeted Syrian chemical weapons facilities in retaliation to a chemical weapons attack in Damascus.
Shocking footage of the chemical attack showed the devastation caused by sarin gas smoke which killed 80 civilians and drew international condemnation.
Assad’s government immediately denied its involvement in a letter to the UN “asserting that Syria has not and will not use toxic gases against its people because it does not have them in the first place”.