In this study, the dosage regimen establishment of cyadox nanosuspension against dairy cow mastitis caused by Staphylococcus aureus (S. aureus) was used as example to provide a general reference for the other novel nanocrystal prepa-rations. The effect of cyadox against S. aureus isolates from dairy cows were firstly estimated and then the dosing regimen of nanosuspension after intramammary administration was optimized according to the model of ex vivo pharmacokinetic (PK) and pharmacodynamic (PD). The therapeutic efficacy of the predicted dosage regimen was evaluated. The results demonstrated that cyadox has a concentration-dependent effect on S. aureus. The smallest and highest values of minimum inhibitory concentration (MIC) against 80 isolates was 8 and 64 μg/mL, respectively. The corresponding MIC50 and MIC90 was 16 and 32 μg/mL, respectively. The MIC against the pathogenic S. aureus SAHZ156001 in broth and milk were 16 and 32 μg/mL, respectively. The AUC0-last and Cmax of cyadox in milk were 4442.877 μg*h/mL and 753.052 μg/mL, respective-ly. According to the inhibitory sigmoid Emax modeling and dosage equation, the daily doses were predicted 1.6, 6.6, and 12.2 mL/gland to achieve bacteriostatic, bactericidal, and elimination effects. The dosage internal was daily administration for continuous three days. The clinical experiment showed that the efficient rates were 100, 100, and 90.9%, and the curative rates were 100, 81.8, and 63.6% in 12.2, 6.6 and 1.6 ml/gland groups, respectively. These results showed that cyadox nano-suspension had a good prospect as intramammary infusion to cure dairy cow mastitis infected by S. aureus. This study will be helpful for providing reference for nanocrystal preparation dosage regimen formulation.