Silver nanoparticles (AgNPs) are widely used in medicine, however, the underlying mechanisms of their action on cellular signaling have not been completely determined, and fundamental studies are required to clarify them. We aimed to investigate AgNPs effects on glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as both the internal control gene and the redox‐sensitive enzyme involved in apoptosis‐related pathways and the formation of amyloid aggregates. To achieve this purpose, MCF‐7 cells were treated with different concentrations (0, 3, 22, and 200 μg/ml) of AgNPs and then cell viability, generation of reactive oxygen species (ROS), induction of apoptosis, expression of GAPDH gene, the formation of amyloid aggregates, and GAPDH activity were assessed. The results indicated that treatment with AgNPs significantly reduced cell viability and increased apoptosis in a dose‐dependent manner. The ROS levels increased at lower concentrations of AgNPs (up to 22 μg/ml) and during short‐term exposure (30 min). The level of GAPDH gene expression was significantly upregulated by 1.22, 1.47, and 1.56 fold, in the concentrations of 3, 22, and 200 μg/ml, respectively. The amount of amyloid aggregates was significantly increased in a dose‐dependent manner. The results of enzyme activity showed that AgNPs were affected on the activity of GAPDH protein, however, it has fluctuated that could not be interpreted by our limited data. In conclusion, our results suggested that AgNPs could affect the GAPDH gene expression and enzyme activity, therefore the selection of GAPDH as a gene and protein internal control in the (AgNPs)‐related studies requires careful consideration. Additionally, AgNPs may cause apoptosis due to the increase in the production of amyloid aggregates.