Although resting-state functional MRI (rs-fMRI) is extensively used to study brain circuitry, recent animal studies suggest a non-neuronal origin of the rs-fMRI signal. We hypothesized that astrocytes may play an important role in rs-fMRI signal. We used chemogenetic technology to selectively activate astrocytes (increasing Ca2+ levels) and recorded rs-fMRI signals in lightly anesthetized rats. Chemogenetic activation of astrocytes following 0.1 mg/kg of clozapine injection induced signal intensity changes and reduced functional connectivity. These in vivo results are consistent with previous brain slice studies, confirming a potentially important role of astrocytes in rs-fMRI signals.