Purpose: To investigate age-related changes in hyaluronan levels in human eyes before and after vitrectomy surgery, we measured the hyaluronan levels in the vitreous samples obtained during vitrectomy and postoperative fluid-air exchange. Methods: We obtained the vitreous during vitrectomy from 26 eyes of 26 patients with macular hole (MH) and 52 eyes of 52 patients with diabetic retinopathy (DR). After vitrectomy, we collected fluid samples during fluid-air exchange from 6 eyes with MH and 9 eyes with DR. The hyaluronan level was measured by the sandwich binding protein assay. Results: In the vitreous of the 54 eyes (26 eyes with MH and 28 eyes with DR and no vitreous hemorrhage), hyaluronan levels significantly decreased with patient age (r = –0.66, p < 0.00000005). Hyaluronan levels in postoperative vitreous fluid were significantly lower in 6 eyes with MH (p < 0.05) and 9 eyes with DR (p < 0.01) than those obtained during vitrectomy. Conclusions: There was a significant decrease in vitreous hyaluronan with aging. High-molecular-weight hyaluronan appears not to be formed in vitreous fluid after vitrectomy.