Skeletal unloading during spaceflight results in bone loss. This study investigated whether laser acupuncture could be an effective countermeasure to prevent unloading-induced bone loss in rats. There were 18 rats that were randomly assigned into three groups: a control group, a tail-suspended group (TS), and a tail-suspended with laser acupuncture treatment group (TSA). The rats in the TSA group were treated with laser acupuncture at the KI1 (Yong Quan) and ST36 (Zu San Li) acupoints of the left leg for 3 min per day. Bone mineral density (BMD), biomechanical properties, and histomorphometry of both tibiae were determined after the animals were euthanized at the end of week 4. Compared with the control group, BMD in the TS group significantly decreased by 12.3% in cortical bone and 15.1% in cancellous bone, whereas BMD in the TSA group decreased by only 3.1% in cortical bone and 9.0% in cancellous bone. The hardness of cortical bone dropped 44.1% in the TS group and 22.3% in the TSA group compared with the control group. The histomorphometry data were in accordance with BMD measurements. Although acupuncture treatment was applied only to the left side, we observed similar changes between the measurements of both the left and right tibiae. Laser acupuncture on KI1 and ST36 can inhibit bone loss in rats subjected to unloading. The fact that similar changes between the right and left sides when only the left limbs were treated suggests that the preventive effect of laser acupuncture occurs via a systemic regulation.