Increased application of silver nanoparticles (AgNPs) has raised concerns on their potential adverse effects on human health. However, the precise toxicological mechanisms are not known in detail. The current study hypothesized that AgNPs induced glycosaminoglycan accumulation in the basement membrane that associated with the up-regulation of its component hyaluronic acid, known as a hydrophilic molecule of binding and retaining water, and caused toxicities in the kidney and cochlea. Rats administered AgNPs through either intravenous or intratympanic injection were observed at different time points after exposure. The concentrations of creatinine and urea in the serum were elevated remarkably, and proteins leaked into the urine were increased. A significant hearing loss over a broad range of frequencies was indicated. AgNP exposure induced glycosaminoglycan accumulation and hyaluronic acid up-regulation in the basement membrane. Abundant apoptotic cell death was demonstrated in the AgNP-exposed organs. Our results suggested that glycosaminoglycan accumulation associated with the up-regulation of hyaluronic acid was involved in the toxicities of kidney and cochlea caused by AgNPs.