In the present study, we aimed to investigate the potential effect of a glucosamine (1,200 mg/day)-based dietary supplement combined with chondroitin sulfate and three antioxidant micronutrients, namely methylsulfonylmethane, guava leaf extract, and vitamin D (test supplement) on osteoarthritis (OA) of the knee. A 16-week, randomized, double-blinded, placebo-controlled trial was conducted involving 32 subjects with symptomatic knee OA. Clinical outcomes were measured using the Japanese Knee Osteoarthritis Measure (JKOM) for symptoms and a study diary-based visual analog scale (diary VAS) for pain at baseline and at weeks 4, 8, 12 and 16 during the 16-week intervention period. Furthermore, biomarkers for cartilage type II collagen degradation (C2C) and synovitis hyaluronan (HA) were measured. As compared with the baseline, the JKOM pain subscale was significantly improved at all of the four assessment time points in the test group, but was not at any time point in the placebo group. On the other hand, all of the four symptom subscales and the aggregated total symptoms were significantly improved in the two groups at one or more time points. However, all of these clinical improvements were greater in extent in the test group than in the placebo group, and there were significant differences between groups in the magnitude of changes from baseline for one subscale 'general activities' and the aggregated total symptoms at week 8 (P<0.05). The results of efficacy assessments with the diary VAS showed that all of the three pain subscales were significantly improved only in the test group at almost all the time points. Moreover, serum levels of C2C and HA were decreased by 10 and 25%, respectively, at week 16 in the test group, albeit not statistically significant, without any detectable changes in the placebo group. In conclusion, although the results obtained in this study were not conclusive, the tested glucosamine-based combination supplement is likely to have a beneficial effect on pain and other symptoms associated with knee OA.