Rouhani asked both Azerbaijan's President Ilham Aliyev and Armenian President Serzh Sargsyan to exercise restraint in dealing with the current conflict in the disputed region.
He also asked the two sides to do all they can to ensure the implementation of a ceasefire currently on hold in Nagorno-Karabakh.
Armenian and Azerbaijani forces said earlier on Wednesday that they were observing a ceasefire recently agreed between the two sides in Nagorno-Karabakh.
The decision came after at least 75 people have reportedly been killed since April 1, when fighting broke out over the region.
“The Islamic Republic of Iran is very upset about the killing of civilians and troops of the two friendly, neighboring countries,” Rouhani said.
He further noted that Iran is ready to use all its capacities for the peaceful settlement of disputes between the two sides through political dialogue.
Rouhani also cautioned that a prolonged conflict over Karabakh would harm the interests of Azerbaijan and Armenia more than any other sides.