Posttraumatic osteoarthritis (PTOA) significantly affects patients with pilon fractures even after adequate anatomical reduction, and treatment strategies targeting the biologic mediators of PTOA are needed. This study was designed to determine the effects of intra-articular injection of platelet-rich plasma (PRP) on synovial fluid (SF) biomarkers for patients undergoing open reduction and internal fixation (ORIF) of pilon fractures. Patients undergoing staged management of pilon fractures were enrolled in a prospective, double-blinded, randomized, and placebo-controlled clinical trial to determine the effects of a single intra-articular injection of leukocyte-reduced PRP on SF biomarkers. Arthrocentesis of the injured and uninjured ankles was performed at the time of external skeletal fixation (ESF) and ORIF. Patients were randomized to receive either autogenous leukocyte-reduced PRP or saline (control) via intra-articular injection into the injured ankle at the time of ESF. SF biomarker concentrations were compared—uninjured, injured pretreatment, and saline-injected or PRP-injected. Eleven patients (PRP, n = 5;​ saline, n = 6) completed the study. Twenty-one uninjured, and 11 injured pretreatment, five PRP-treated, and six saline-treated SF samples were analyzed. PRP-treated SF contained significantly higher levels of PDGF-AA (p = 0.046) and significantly lower levels of MMP-3 (p = 0.042), MMP-9 (p = 0.009), IL-1β (p = 0.049), IL-6 (p < 0.01), IL-8 (p = 0.048), and PGE2 (p < 0.04). This study provided mechanistic data to suggest that a single intraarticular injection of leukocyte-reduced PRP is associated with anti-inflammatory, anti-degradative, and anabolic responses compared with saline control. These findings provide the impetus for investigating long-term clinical outcomes after PRP injection as an orthobiologic adjunct to ORIF for mitigating the incidence and severity of PTOA after pilon fractures.