Background: To characterize the environment of deep underground laboratory (DUGL) with a rock cover of 1470m and observe the effect of the DUGL environment on the growth and metabolism of Chinese hamster V79 cells. Results: Six environmental parameters in the DUGL and an above ground laboratory (AGL; control) were monitored. Compared to the AGL, O2 concentration was not significantly different, total Îł ray dose rate was significantly lower (p=0.005), and relative humidity (p<0.001), air pressure (p<0.001), and concentration of CO2 and radon gas (p<0.001) were significantly higher in the DUGL. The growth curves of cultured V79 cells showed cell proliferation was slower in the DUGL. Tandem mass tag (TMT) proteomics analysis was performed to identify differentially abundant proteins (DAPs) in V79 cells cultured in the DUGL and AGL. Parallel Reaction Monitoring (PRM) was conducted to verify TMT results. TMT detected 980 DAPs, defined as proteins with a â„1.2-absolute fold change in relative abundance (p <0.05) between V79 cells cultured in the DUGL and AGL. Of these, 576 proteins were up-regulated and 404 proteins were down-regulated in V79 cells cultured in the DUGL. GO term enrichment analysis of up-regulated proteins revealed enrichment of proteins involved in translation, ribosome, proton-transporting ATP synthase activity, oxygen binding, and oxygen transporter activity et al. GO term enrichment analysis of downregulated proteins demonstrated enrichment of proteins involved in the endoplasmic reticulum lumenand respiratory chain. KEGG pathway analysis revealed that ribosome (p<0.001), base excision repair (p<0.001), RNA transport (p=0.009), Huntington's disease (p=0.023), and oxidative phosphorylation (OXPPL) (p=0.035) pathways were significantly enriched. Conclusion: Proliferation of V79 cells was inhibited in the DUGL, likely because cells were exposed to reduced cosmic ray muons flux. There were apparent changes in the proteome profile of the V79 cells cultured in the DUGL, which affected proteins related to the ribosome, RNA transport, translation, energy metabolism, and DNA repair.These proteins may have induced cellular changes that delayed proliferation but enhanced survival, making the V79 cells adaptable to the changing environment. Our findings provide insight into the cellular stress response that is triggered in the absence of normal levels of radiation.