Despite numerous investigations into AgNP-induced toxicity, little has been taken into consideration the potential health impacts of aged AgNPs in comparison to fresh AgNPs. In the current study, we scrutinized the potential effects of aged AgNPs in animals. We first found that AgNPs underwent morphological transformations after natural ageing in aqueous solution upon exposure to air and sunlight for 9 days, as characterized by significant aggregation with increase of particle size approximately by 2 fold. Meanwhile, dissolved Ag ions from aged AgNPs increased by 33% compared to fresh AgNPs. Strikingly, the acute exposure results showed that aged AgNPs induced lower toxicity in mice relative to fresh AgNPs. Aged AgNPs caused milder local inflammation in the peritoneal cavity of mice, as evidenced by 63% reduction of tumor necrosis factor α (TNF-α) than that induced by fresh AgNPs. The deposition mass of aged AgNPs in the liver, spleen, lung and kidney was diminished by 69%, 39%, 83% and 40%, respectively, compared to the distribution profiles in response to fresh AgNPs. Whereby, milder splenic hyperemia was observed, and no significant hepatoxicity was found. Additionally, aged AgNPs provoked milder increase of periphery leukocytes and malondialdehyde (MDA) in mice in comparison to fresh AgNPs. Taken together, this study unraveled that the ageing process elicited remarkable alterations to physicochemical properties and toxic effects as well. This study would provide new insights into the potential health impacts of AgNPs under transformation-determined exposure scenarios.