Non-invasive photobiomodulation therapy (PBMT), employing specific infrared light wavelengths to stimulate biological tissues, has recently gained attention for its application to treat neurological disorders. Here, we aimed to uncover the cellular targets of PBMT and assess its potential as a therapeutic intervention for multiple sclerosis (MS). We applied daily dorsoventral PBMT in an experimental autoimmune encephalomyelitis (EAE) mouse model, which recapitulates key features of MS, and revealed a strong positive impact of PBMT on the sensorimotor deficits. To understand the cellular mechanisms underlying these striking effects, we used state-of-the-art tools and methods ranging from two-photon longitudinal imaging of triple fluorescent reporter mice to histological investigations and patch-clamp electrophysiological recordings. We found that PBMT induced anti-inflammatory and neuroprotective effects in the dorsal spinal cord. PBMT prevented peripheral immune cell infiltration, glial reactivity, as well as the EAE-induced hyperexcitability of spinal interneurons, both in dorsal and ventral areas, which likely underlies the behavioral effects of the treatment. Thus, aside from confirming the safety of PBMT in healthy mice, our preclinical investigation suggests that PBMT exerts a systemic and beneficial effect on the physiopathology of EAE, primarily resulting in the modulation of the inflammatory processes. PBMT may therefore represent a new valuable therapeutic option to treat MS symptoms.