The centre-left Bratusek lost a vote of support by members of her Positive Slovenia (PS) after a challenge by party founder and Ljubljana mayor Zoran Jankovic early today.
Bratusek, 44, said ahead of the vote she would resign if this happened. It was unclear early today what her next step would be.
"Without the support of my own party, I can no longer be the head of government, I cannot be on equal footing with the other (coalition) parties," a visibly emotional Bratusek told PS members ahead of the vote.
Under Jankovic, a charismatic supermarket millionnaire, the PS came first in elections in December 2011 but he failed to form a coalition and he stepped down as party leader in early 2013 following corruption allegations.
Once a model newcomer to the European Union and the eurozone, the former Yugoslav republic of two million people, and in particular its banks, was hit hard by the global financial disaster of 2008-9 and the subsequent eurozone debt crisis.
But in 13 months in office, Bratusek has managed to satisfy Brussels that Slovenia does not need a bailout by pumping more capital into the banks and with a programme of privatisations and reforms.