BACKGROUND: Periodontitis is a prevalent oral disease that significantly impacts human quality of life. This study aimed at the influence of microRNA-299-5p (miR-299-5p) on regulating lipopolysaccharide (LPS)-induced osteogenic differentiation of periodontal ligament stem cells (PDLSCs). METHODS: The 105 periodontitis patients and 102 healthy periodontal volunteers (HC) were recruited with their clinical baseline data. miR-299-5p expression in saliva was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To identify the target relationship between miR-299-5p and pumilio RNA-binding family member 2 (PUM2), the dual luciferase reporter gene was explored. PDLSCs were treated with LPS and subjected to osteogenic differentiation induction. The secretion of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) was determined by enzyme-linked immunosorbent assay. mRNA expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN) was detected by qRT-PCR. RESULTS: miR-299-5p expression was markedly reduced in periodontitis patients' saliva and negatively correlated with patients' attachment loss, bleeding index, plaque index, probing pocket depth, and TNF-α, IL-6, and IL-1β levels. miR-299-5p could be well distinguished between periodontitis and HC. LPS induction dramatically stimulated the secretion of inflammatory factors and inhibited the differentiation of PDLSCs, which was counteracted by miR-299-5p overexpression. miR-299-5p was detected to target PUM2, which could exacerbate the inflammatory condition of PDLSCs and lead to differentiation defects. CONCLUSIONS: miR-299-5p has emerged as a promising diagnostic marker for periodontitis and may attenuate inflammation and contribute to osteogenic differentiation in PDLSCs cells by targeting PUM2.