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Radiographic severity of OA was determined by Kellgren-Lawrence (K-L) grades. PACAP levels were measured by ELISA before treatment, and 4 and 8 wk following hyaluronic acid (HA) injection. Levels of IL-1 and MMP-3 were also detected. The numeric pain scale (NPS), revised Oxford Knee Score (OKS), and American Knee Society Score (AKSS) were employed to evaluate to symptomatic severity. Receiver-operating-characteristic (ROC) curve analysis was carried out to compare the diagnostic value of PACAP, IL-1, and MMP-3 for the K-L grade. PACAP concentrations in SF but not serum were significantly lower in OA patients compared with controls. SF PACAP levels were negatively associated with K-L grades and higher NPS as well as worse AKSS and OKS. Further analysis demonstrated that PACAP concentration in SF was negatively correlated with expressions of IL-1 as well as MMP-3 and may act as a marker for radiographic progression along with MMP-3. Last, we found SF PACAP levels exhibited an incremental trend after HA injection. 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