Thirdhand smoke (THS), a residual cigarette smoke still remaining in indoor environment long after active smoking has ceased, has aroused more and more people's attention on health. However, the impact of THS exposure on the male reproductive system, particularly in in-vivo study, remain unclear. Thus, this study aims to investigate the adverse effects of 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA), a special component in THS, on testis. Herein, adult ICR mice were orally administered diluted dimethylsulfoxide (control), 0.075 μg/kg and 0.15 μg/kg NNA for 10 weeks. The findings revealed that NNA exposure, especially in 0.15 μg/kg dosage, significantly reduced the epithelium height and tubular area within the testis. Transcriptomic analysis indicated that NNA exposure interfered with the expression of genes associated with oxygen transport and oxygen binding processes, such as Hbb-bs, the downregulation of which also led to oxidative damage. Additionally, NNA exposure induced oxidative damage in testis by decreased levels of SOD and GSH enzymes, along with an increase in MDA level. In summary, our results demonstrate that NNA exposure causes impaired testicular structure and oxidative damage, indicating its testicular toxicity. This finding not only implies the detrimental effect of NNA on male reproduction, but also provides a fundamental basis for further research on the impact of THS exposure on male reproductive health.