Central sensitization driven by glial activation-mediated neuroinflammation is recognized as a key mechanism in pain processing. Laser moxibustion using low-intensity laser irradiation of corresponding acupoints significantly relieves knee osteoarthritis (KOA) pain. However, the underlying mechanism of its effects on KOA pain is still not completely understood. In this study, we aimed to investigate whether laser moxibustion could alleviate KOA pain by inhibiting spinal glial activation and proinflammatory cytokines upregulation in monosodium iodoacetate (MIA)-induced KOA pain in rats. Sprague-Dawley rats were divided randomly into three groups: Saline + Sham Laser, MIA + Laser, and MIA + Sham Laser. A 10.6 μm laser was used to irradiate ST35 (Dubi) for 10 min once every 2 days for a total of seven applications. The paw withdrawal mechanical threshold and weight-bearing distribution were performed to evaluate the nociceptive behaviors. Spinal expressions of microglial marker, ionized calcium binding adaptor molecule-1 (Iba-1); astrocyte marker, glial fibrillary acidic protein (GFAP); pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were measured 14 days after MIA injection. The results showed that laser moxibustion significantly reversed the MIA-induced mechanical hyperalgesia and weight-bearing difference up to 14 days compared with MIA + Sham Laser group ( < 0.05 or < 0.01). Moreover, both the protein level and immunofluorescence intensity of Iba-1 in the ipsilateral spinal cord dorsal horn were markedly decreased in the MIA + Laser group than those in the MIA + Sham Laser group ( < 0.01). However, there was no significant difference in the expression of GFAP between groups ( > 0.05). In addition, laser moxibustion decreased the upregulation of TNF-α, IL-1β, and IL-6 compared with the MIA + Sham Laser group ( < 0.01). This study demonstrated that laser moxibustion at ST35 significantly alleviated MIA-induced KOA pain through inhibition of the microglial activation-mediated neuroinflammation, at least partially, by suppressing the production of proinflammatory cytokines, which may provide a potential analgesic target for KOA pain relief.