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MicroRNA-335-5p has been reported to regulate osteogenic and chondrogenic differentiations of mesenchymal stem cells. The aim of this study was to explore the function and regulation mechanism of miR-335-5p in apical periodontitis (AP).Total RNAs were extracted from human periodontal ligament fibroblasts (HPDLFs), 10 AP tissues, and 6 healthy periodontal ligament tissues using lysis buffer. Gene expression was detected using real-time polymerase chain reaction. The Dual Luciferase Assay (Promega, Madison, WI) was used to test miR-335-5p directly targeted urokinase-type plasminogen activator receptor (uPAR) and the receptor activator of nuclear factor kappa-B ligand (RANKL). Western Blot was used to detect protein expressions of RANKL, uPAR, and the fragile X–related 1 gene (FXR1). The enzyme-linked immunosorbent assay was used to detect the secretions of interleukin 6, tumor necrosis factor alpha, and RANKL. Data were analyzed using the Student t test.miR-335-5p acted as a positive mediator in HPDLF inflammation (P < .05). Two targets of miR-335-5p, uPAR and RANKL, were identified. Interestingly, uPAR was repressed by miR-335-5p at the basal level, but it can be relieved from miR-335-5p–mediated repression, which is called derepression, when HPDLFs were subjected to lipopolysaccharide stimulation. miR-335-5p promoted RANKL in HPDLFs regardless of whether or not it was under inflammatory conditions (P < .05). We proved FXR1 was responsible for the derepression of uPAR from miR-335-5p (P < .01). Both FXR1 and uPAR were positive mediators in HPDLF inflammation (P < .05). miR-335-5p, uPAR, RANKL, and FXR1 had the same expression profiles in HPDLF inflammation and AP tissues (P < .05).Our data showed that miR-335-5p may play dual roles in AP, and it might be considered as a target for therapeutic potency in clinical applications.miR-335-5p acted as a positive regulator in the inflammatory response of human periodontal ligament fibroblasts (HPDLFs).As 1 target of miR-335-5p, the urokinase-type plasminogen activator receptor (uPAR) switched from being repressed to being activated under inflammatory conditions.miR-335-5p directly promoted receptor activator of nuclear factor kappa-B ligand (RANKL) in HPDLFs both at basal level and LPS-stimulating level.FXR1 is involved in the relief of uPAR from miR-335-5p–mediated repression in HPDLF inflammation.