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Abstract. Cutibacterium acnes isolation from spine tissue can be challenging because the organism can represent a contaminant. There is a paucity of data regarding the role of C. acnes in non-hardware-associated vertebral osteomyelitis (VO). Herein we evaluate the clinical and microbiological characteristics, treatment, and outcome of patients with C. acnes VO. Data were retrospectively collected from adults with a positive spine culture for C. acnes at Mayo Clinic, Rochester (MN), from 2011 to 2021. Patients with spinal hardware and polymicrobial infections were excluded. Of the subjects, 16 showed radiological and clinical findings of VO: 87.5 % were male, the average age was 58 years (±15 SD), and back pain was the predominant symptom. Of the lesions, 89.5 % involved the thoracic spine. Of the subjects, 69 % had experienced an antecedent event at the site of VO. In five subjects, C. acnes was isolated after 7 d of anaerobic culture incubation. Thirteen subjects were treated with parenteral β-lactams, and three with oral antimicrobials, without any evidence of recurrence. Twenty-one subjects were not treated for VO, as C. acnes was considered a contaminant; at follow-up, none had evidence of progressive disease. C. acnes should be part of microbiological differential diagnosis in patients with suspected VO, especially in the context of a prior spinal procedure. Anaerobic spine cultures should undergo prolonged incubation to enable recovery of C. acnes. C. acnes VO may be managed with oral or parenteral antimicrobial therapy. Without clinical and radiological evidence of VO, a single positive culture of C. acnes from spine tissue frequently represents contaminants.