Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions (Ag⁺) on the proliferation of human skin keratinocytes (HaCaT) and the production of intracellular reactive oxygen species (ROS). After treating HaCaT cells with Ag⁺and/or the active oxygen scavenger N-acetyl cysteine (NAC), cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine (BrdU) incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen (PCNA) immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag⁺ concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10⁻⁶ and 10⁻⁵ mol/L Ag⁺ at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5–60 min after exposure to Ag⁺. The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10⁻⁶ and 10⁻⁵ mol/L Ag⁺, with 10⁻⁵ mol/L Ag⁺ markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag⁺ concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag⁺ concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.