Heat shock protein (Hsp) 60 has been implicated in the pathogenesis of various inflammatory and autoimmune diseases. This study aimed to investigate synovial fluid and serum concentrations of Hsp60 and anti-Hsp60 and their relationship with juvenile idiopathic arthritis (JIA). Forty-eight patients with JIA, including 22 oligo-articular, 19 poly-articular, and 7 systemic diseases, and 33 normal controls were enrolled in this study. Synovial fluid and serum Hsp60 and anti-Hsp60 concentrations were measured via ELISA. Serum concentrations of Hsp60 of active and inactive oligo- and poly-articular JIA were significantly higher than those of normal controls. Serum concentration of anti-Hsp60 in active oligo-articular JIA was higher than that of normal controls (49.25 vs. 35.76 ng/mL, p = 0.059). Similarly, serum concentration of anti-Hsp60 in active poly-articular JIA was significantly higher than that of inactive samples (65.05 vs. 26.54 ng/mL, p = 0.008). In addition, serum concentration of Hsp60 correlated with the time required for remission from flare-ups in patients with JIA. Serum concentration of Hsp60 correlated well with time required for remission from flare-ups in patients with JIA, representing a potential disease marker to monitor disease activity.