Sports-related tendon injuries are commonly treated with nonsteroidal antiinflammatory drugs. This study was designed to determine the in vitro effect of ibuprofen on the proliferation of tendon cells intrinsic to rat Achilles tendon. Furthermore, the existence of a correlation between this effect and the expression of the cyclin kinase inhibitor p21(CIP1) and retinoblastoma (Rb) protein was also examined. Using cultured tendon cells, cell viability was evaluated by MTT assay. To determine whether apoptosis was related to the effect of ibuprofen, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay was used. The mitotic index (MI) was calculated from the number of cells in the mitotic phase as stained and identified by propidium iodide. The mRNA expression of p21(CIP1) was determined by reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions of p21(CIP1) and Rb protein were determined by Western blot analysis. A dose-dependent decrease in the cellularity of tendon cells by ibuprofen was demonstrated by MTT assay (p<0.001). However, TUNEL assay revealed no evidence of apoptosis. Ibuprofen dose-dependently reduced the MI (p<0.001). Upregulation of p21(CIP1) both at the levels of mRNA expression and protein was revealed from RT-PCR and Western blot analyses. The inhibition of Rb protein phosphorylation was also noted in ibuprofen-treated cells. In conclusion, ibuprofen inhibits tendon cell proliferation in a process that is probably mediated by the upregulation of p21(CIP1) and reduced phosphorylation of Rb protein.