Aim To observe the effect of oxidative stress on nucleolar impairment in C₂C₁₂ and clarify the possible molecular mechanism. Methods 0.5 mmol/L peroxide hydrogen (H 2O 2) were added into the cultured C₂C₁₂ cell lines to mimic oxidative stress. Toluidine Blue staining and total protein synthesis analysis were performed to assess H 2O 2-induced nucleolar structural and functional injury respectively. Immunoblotting and antisense oligonucleotide were used to detect the changes of nucleolar protein nucleolin (also named C23). Results Toluidine Blue staining showed predominantly compact, centrally localized nucleoli in intact control cells, but in H 2O 2-treated cells, an early onset of nucleolar segregation could be found after 3 h and 6 h. Total protein synthesis analysis revealed that compared with normal cells, the capability of cellular protein synthesis is significantly decreased after 6 h treatment with H 2O 2 (p<0.05), and lasted for 24 h. Moreover, an 80 kDa band of nucleolin was found by using immunoblotting after 1 h treatment with H 2O 2, and accompanied by down-regulation of its primary 110 kDa band. The 110 kDa band decreased remarkably after 6 h and 12 h treatment with H 2O 2. After transfected nucleolin antisense oligonucleotides for 24 h and 48 h, expression of nucleolin was down- regulated significantly, and nucleolar segregation and inhibition of total protein synthesis could be observed. Conclusion The molecular mechanism of oxidative stress-mediated nucleolar impairment is related to cleavage and down-regulation of intact nucleolin.