Nucleus pulposus (NP) cell senescence is involved in disc degeneration. The in situ osmolarity within the NP region is an important regulator of disc cell’s biology. However, its effects on NP cell senescence remain unclear. The present study was aimed to investigate the effects and mechanism of hyper-osmolarity on NP cell senescence. Rat NP cells were cultured in the in situ -osmolarity medium and hyper-osmolarity medium. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was added along with the medium to investigate the role of oxidative injury. Cell cycle, cell proliferation, senescence associated β-galactosidase (SA-β-Gal) activity, telomerase activity, expression of senescence markers (p16 and p53) and matrix molecules (aggrecan and collagen II) were tested to assess NP cell senescence. Compared with the in situ -osmolarity culture, hyper-osmolarity culture significantly decreased cell proliferation and telomerase activity, increased SA-β-Gal activity and cell fraction in the G 0 /G 1 phase, up-regulated expression of senescence markers (p16 and p53) and down-regulated expression of matrix molecules (aggrecan and collagen II), and increased intracellular ROS accumulation. However, addition of NAC partly reversed these effects of hyper-osmolarity culture on cellular senescence and decreased ROS content in NP cells. In conclusion, a hyper-osmolarity culture promotes NP cell senescence through inducing oxidative stress injury. The present study provides new knowledge on NP cell senescence and helps us to better understand the mechanism of disc degeneration.