ABSTRACT Previous results showed that intermittently administered irisin improves bone mass in normal mice and prevents the development of disuse-induced osteoporosis and muscular atrophy in hindlimb-suspended mice, a murine model able to mimic the absence of mechanical loading. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/β5 receptors. To better understand the action of irisin on these cells, we investigated the downstream signaling cascades in osteocyte-like cells (MLO-Y4) treated with recombinant irisin (rec-irisin) in vitro and we analyzed survival of osteocytes and caspase activation in cortical bone of osteoporotic mice treated with rec-irisin in vivo. Our results revealed that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 (2.5-fold, p < .05) through an Erk-dependent pathway in osteocytes. Some key genes expressed by MLO-Y4 cells were modulated by long-term irisin treatment, either continuously administered or given with intermittent short pulses. Interestingly, Sost mRNA was severely downregulated only upon intermittent irisin administration (10-fold, p < .001). Furthermore, rec-irisin upregulated Tfam mRNA (fourfold, p < .05) and Bcl2/Bax ratio (twofold, p < .05) in MLO-Y4 cells. By detecting caspase-9 and caspase-3, we also found that rec-irisin inhibited apoptosis induced by hydrogen peroxide and dexamethasone, respectively. In cortical bone of unloading C57BL6 mice treated with vehicle (unload-veh), irisin prevented disuse-induced reduction of viable osteocytes (+30% versus unload-veh, p < .05) and increase of empty lacunae (+110% versus unload-veh, p < .05), as well as caspase-9 (threefold, p < .05) and caspase-3 (twofold, p < .05) activations. Our findings revealed underlying mechanisms of irisin action on osteocytes, which increases their functions and exerts anti-apoptotic effects, confirming that mechanosensor cells of bone are sensitive to the exercise-mimetic myokine irisin. © 2019 American Society for Bone and Mineral Research. Abstract Schematic representation of irisin's action on osteocytes to activate ERK phosphorylation, which in turn stimulates the increase of Atf4 mRNA levels. A recent study showed that irisin increases survival of osteocytes acting through integrin αV/β5 receptors. To elucidate the action of irisin on osteocytes, we investigated the downstream signaling cascades in MLO-Y4 cells treated with recombinant irisin (rec-irisin). We found that rec-irisin activated the MAP kinases Erk1 and Erk2 and increased the expression of the transcription factor Atf4 through an Erk-dependent pathway in osteocytes.