We examined the effects of ~30 days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3b content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3b was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight. This study derives results from the Western blot assay using tibialis anterior tissue from the RR9 mission. The tibialis anterior data in this study are related to other studies using tissues from the same experiment; OSD-654 (tibia), OSD-661 (lumbar spine), OSD-662 (soleus), OSD-663 (femur), and OSD-664 (extensor digitorum longus).