Background Astrocytes are abundant in the brain and their calcium signaling is reported to have an important effect on neuronal activity in both physiological and pathological conditions. Low frequency focused ultrasound (FUS) has recently emerged as a powerful noninvasive neuromodulation approach, yet its impact on astrocyte calcium dynamics in different brain states in vivo is poorly understood. Objective This study aimed to elucidate the effects of non-thermal FUS on astrocyte calcium dynamic with in vivo cellular resolution and cell type specific recording and identify whether the influences of FUS on cortical astrocytes and neurons are distinctive and state dependent. Methods Here we combined a customized 0.521MHz FUS transducer with two-photon microscopy, allowing simultaneous single-cell resolution imaging and FUS stimulation at intensities of 0.91 or 1.5 W/cm2 to examine astrocyte and neuronal calcium responses in somatosensory cortex of both awake and lightly anesthetized mice. Functional clustering analysis was performed to identify calcium response activated or inhibited subpopulations. Results In awake mice, FUS significantly enhanced the amplitude, frequency, and temporal integral of astrocyte calcium transients, while suppressing neuronal calcium activity and reducing the proportion of activated neuronal subpopulations. In contrast, lightly anesthetized mice displayed a blunted yet increased astrocyte response and negligible neuronal modulation under FUS, suggesting that baseline suppression from anesthesia partially masks FUS effects. Conclusions Our study demonstrated that FUS elicited distinctive, state-dependent effects on cortical astrocytes and neurons, highlighting astrocytes as previously underappreciated targets of ultrasound neuromodulation. These findings will pave the way for FUS based therapies targeting astrocyte neuron interactions in conditions involving abnormal brain excitability.