Non-specific low back pain (LBP) is responsible for triggering increased biomarkers levels. In this way, photobiomodulation therapy (PBMT) may be an interesting alternative to treat these patients. One of the possible biological mechanisms of PBMT involved to decrease pain intensity in patients with musculoskeletal disorders is modulation of the inflammatory mediators' levels. The aim of this study was to evaluate the effects of PBMT compared with placebo on inflammatory mediators' levels and pain intensity in patients with chronic non-specific LBP. A prospectively registered, randomized triple-blinded (volunteers, therapists, and assessors), placebo-controlled trial was performed. Eighteen patients with chronic non-specific LBP were recruited and treated with a single session of active PBMT or placebo PBMT. The primary outcome of the study was serum prostaglandin E levels and the secondary outcomes were tumor necrosis factor-α, interleukin-6 levels, and pain intensity. All outcomes were measured before and after 15 minutes of treatment session. PBMT was able to decrease prostaglandin E levels at post-treatment compared with placebo, with a mean difference of -1470 pg/ml, 95% confidence interval -2906 to -33.67 in patients with LBP. There was no difference between groups in the other measured outcomes. Patients did not report any adverse events. Our results suggest that PBMT was able to modulate prostaglandin E levels, indicating that this may be one of the mechanisms involved in the analgesic effects of PBMT in patients with LBP. Trial registration number (ClinicalTrials.gov): NCT03859505. Lasers Surg. Med. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals, Inc.