Ayotte's defeat to Governor Maggie Hassan in New Hampshire means another pick-up for the Democrats in the battle for control of the Senate.
But Republicans outperformed expectations in yesterday's election and kept their grip on the chamber, with a likely final tally of 52 Republicans to 48 Democrats.
The two New Hampshire rivals were separated by just 600 votes by mid-afternoon today, some 22 hours after polls closed in the US election that saw Republican presidential nominee Trump deliver a stunning defeat of Democrat Hillary Clinton.
Republican lawmakers had debated their strategy in recent months given the toxic rhetoric emanating from Trump, who had called Mexicans rapists, demeaned women and vowed to ban Muslims from entering the United States.
Some, particularly in swing states, strategised about whether to endorse Trump and incur the wrath of angry independent voters, or reject him and risk a backlash from his supporters.
After last month's release of a 2005 video clip that caught Trump making vulgar remarks about women, Ayotte and Joe Heck, a congressman who lost his Senate bid Tuesday in Nevada, were among the first Republican lawmakers to reject the party's presidential nominee.