Zinc oxide nanoparticles (ZnO NPs) are being used in a wide range of applications including industry, commercial products and medicine field. Numerous mechanistic studies for ZnO NPs’ toxicity are performed on pristine (fresh) NPs. However, the cytotoxicity induced by the transformed (aged) ZnO NPs and the underlying mechanisms remain unclear. Here, we firstly confirmed the physicochemical transformation of ZnO NPs underwent over time and compared the cytotoxicity induced by fresh and aged NPs. Then, we found that fresh NPs induced higher apoptosis levelthan aged NPs. Accordingly, RNA sequencing data from aged ZnO NP-treated human-hamster hybrid (AL) cells showed that p53, PI3k-Akt, FoXO, Glutathione, ErbB, HIF-1, Oxytocin and Jak-STAT signaling pathway were enriched but no apoptosis pathway. Quantitative PCR results confirmed the significantly higher mRNA level of IL1B and CD69 in fresh NP-treated groups compared to that of aged ZnO NP- and zinc chloride-treated groups. Our data indicated that the lower cytotoxicity of aged ZnO NPs is closely related to the low level of apoptosis induced by it and that the transcriptional regulation of the multiple pathways activated by aged NPs helps to build the cellular homeostasis. Our results highlight the aging (environmental transformation) process to the toxicity and safety assessment of ZnO NPs.