The purpose of the study was to investigate the pharmacokinetics of cefquinome in plasma and milk samples of lactating Chinese Holstein following a single intramammary administration into one quarter at the dose of 75 mg. Residue depletion of cefquinome in milk administrated at one quarter following three consecutive infusions at the same dose were also carried out. Cefquinome concentrations in plasma and milk were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. A non-compartmental analysis was used to obtain the pharmacokinetic parameters of cefquinome. Following the single treatment, cefquinome wasn’t detected in any of the plasma samples. The concentration of cefquinome in milk reached peaked values (Cmax) of (599.00±322.00) μg mL-1 at 2 h after administration (Tmax), elimination half-life (t1/2λz) was (4.63±0.26) h, area under the concentration-time curve (AUC0-∞) was (4 890.19±1 906.98) μg mL-1 h, and mean residence time (MRT) was (6.03±2.27) h. In residue depletion study, cefquinome concentrations in 5 out of 6 milk samples at 72 h were lower than the maximum residue limit ifxed by the European regulatory agency (20μg kg-1 for cefquinome) and cefquinome still could be detected in milk of treated quarters at 120 h post-treatment. The maximum concentration (Cmax) of cefquinome in milk from treated quarters was (486.50±262.92) μg mL-1 and arrived at 6 h after administration (Tmax), elimination half-life (t1/2λz) was (6.30±0.76) h, area under the concentration-time curve (AUC0-∞) was (44 747.79±11 434.43) μg mL-1 h, and mean residence time (MRT) was (10.09±1.40) h. This study showed that cefquinome has the feature of poor penetration into blood and was eliminated quickly from milk in lactating cows after intramammary administration.