The aim of this study was to investigate the effect of hyaluronic acid (HA) on the expression of osteopontin (OPN) and CD44 mRNA of fibroblast-like synoviocytes (FLS) in the patients with osteoarthritis (OA) of the knee. FLS were obtained from the knees of 10 patients with OA. Real-time quantitative polymerase chain reaction (Q-PCR) was used to assess the expression of the OPN mRNA and CD44 mRNA. The relative OPN mRNA expression in HA group (6.47 ± 2.30-fold) was significantly higher than in the control group (P = 0.045, P < 0.05), while in HYL group (0.65 ± 0.21-fold) it was lower than in the control group (P = 0.037, P < 0.05), and the difference in OPN mRNA expression between HA group and HYL group also showed statistically significant (P = 0.001, P < 0.05); however, there was no significant difference between each group of the relative CD44 mRNA expression (P > 0.05). Our study showed that HA can upregulate OPN mRNA expression in OA fibroblast-like synoviocytes, and the high expression of OPN mRNA in OA may be a result of increased HA level of OA synovitis; however, HA cannot affect the CD44 mRNA expression in OA fibroblast-like synoviocytes, and the high expression of CD44 mRNA in OA may be not a result of increased HA level of OA synovitis.