LIPUS, low intensity pulsed ultrasounds, are considered a safe and non-invasive tool that have been used extensively in medicine for chronic diseases. We evaluated the effects produced by LIPUS on the physiological behaviour of mouse and human adipose stem cells (ASCs) as well as on adipose tissue explants. LIPUS stimulation for one minute did not affect mouse or human ASC proliferation or apoptosis, with no evident changes in morphology or cell growth. Further, RNA-seq analysis revealed more than 200 genes differentially expressed after ultrasound stimulation of mouse and human ASCs. Among them, the changes in gene expression mainly belong to the canonical pathways of stemness, energy metabolism or chemokine signalling. On the other hand, the migration ability of mouse and human ASCs was partially affected by the ultrasound protocol, slightly increasing their capacity to migrate, while maintaining their stemness properties. Major affection was detected on the adipocyte differentiation capacity. Indeed, LIPUS-treated ASCs were unable to differentiate into mature adipocytes and their inflammatory secretion profile was reduced in both mouse and human ASCs. These effects were confirmed on an ex vivo model of adipose tissue explants, demonstrating that LIPUS also provoked an anti-inflammatory profile in the adipose tissue while reducing adipocytes differentiation and lipids content. Overall, the strong adipogenesis blockade, the reduction of their secretion profile and the RNA-seq analysis suggest that LIPUS-treated ASCs may be prevented from differentiating into mature adipocytes and from exacerbating inflammation, thus limiting their contribution to obesity.