Ciprofloxacin-induced tendinopathy and tendon rupture have been previously described, principally affecting the Achilles tendon. However, the underlying mechanism remains unclear. Tendon healing requires migration of tenocytes to the repair site, followed by proliferation and synthesis of the extracellular matrix. This study was designed to determine the effect of ciprofloxacin on migration of tenocytes intrinsic to rat Achilles tendon. Whether a correlation exits between this effect and the expression and phosphorylation of focal adhesion kinase (FAK), which is a positive regulator of cell motility, was also investigated. Using cultured tenocytes, migration was evaluated by counting the number of initial cell outgrowth from the tendon explants and by transwell filter migration assay. Tenocyte spreading was also evaluated microscopically. The serum-induced protein expression and phosphorylation of FAK were determined by Western blot analysis in synchronized tenocytes. Ciprofloxacin dose-dependently inhibited tenocytes outgrowth from the explants ex vivo, migration of tenocytes through the transwell filter, as well as cell spreading in vitro. Suppression of FAK phosphorylation was revealed by Western blot analyses. In conclusion, ciprofloxacin inhibits tenocyte migration in a process that is probably mediated by inhibition of FAK phosphorylation.