It was sharp criticism from an incumbent who had previously insisted he would support the GOP nominee.
The Illinois Republican, who faces a tough challenge this November from Illinois Democratic Rep. Tammy Duckworth, tweeted Tuesday that based on his background in the military, he does not have faith in Trump to lead.
"Given my military experience, Donald Trump does not have the temperament to command our military or our nuclear arsenal," Kirk wrote.
Kirk, who told CNN that he would write in former CIA director David Petraeus this fall, followed up with a statement saying he objects to Trump's recent comments about a federal judge's Mexican heritage.
"I find Donald Trump's belief that an American-born judge of Mexican descent is incapable of fairly presiding over his case is not only dead wrong, it is un-American," Kirk said.
Trump said in a statement Tuesday that his comments had been "misconstrued" by the media.
"I am friends with and employ thousands of people of Mexican and Hispanic descent," Trump said in his statement. "The American justice system relies on fair and impartial judges. All judges should be held to that standard. I do not feel that one's heritage makes them incapable of being impartial, but, based on the rulings that I have received in the Trump University civil case, I feel justified in questioning whether I am receiving a fair trial," Trump said in a statement his campaign released Tuesday afternoon."
Kirk added in his statement that he had hoped Trump would tone down his rhetoric.
"While I oppose the Democratic nominee, Donald Trump's latest statements, in context with past attacks on Hispanics, women and the disabled like me, make it certain that I cannot and will not support my party's nominee for President regardless of the political impact on my candidacy or the Republican Party," Kirk said.
The rebuke of Trump was a departure from Kirk's earlier position on the mogul. Before Trump had solidified his place as the presumptive nominee, Kirk told CNN that he would "certainly" back Trump if he were the party's nominee and even said running alongside Trump on the ticket might be beneficial to his race.
Kirk's Democratic opponent was quick to jump on him for the tweet. Duckworth, an Iraq War veteran who lost her legs in conflict, spun Kirk's tweet back around.
"Given my military experience, Mark Kirk doesn't have the temperament to be our Senator," Duckworth tweeted
, linking to a new campaign site that links Kirk to Trump with a quiz asking who said which quote.
Kirk served in the Navy Reserves for 24 years.