6-Hydroxykaempferol-3,6-diglucoside is an important flavonoid constituent of Carthamus tinctorius L., with potential antioxidant and myocardial protective effects. However, its pharmacokinetic characteristics in normal and diabetic cardiomyopathy (DCM) mice remain unknown. This study was designed to explore the pharmacokinetic differences of this component between normal mice and DCM mice induced by a high-fat diet combined with streptozotocin. All mice were intragastrically administered at a dose of 20 mg/kg. Plasma samples were collected at different time points, and the plasma concentration was determined by LC-MS/MS. Compared with normal mice, DCM mice showed significantly decreased AUC0-t and Cmax, indicating reduced absorption and bioavailability, while plasma clearance was markedly increased, suggesting accelerated elimination. No significant differences in Tmax and t1/2 were observed. In contrast to our previous study, another core safflower component, HSYA, exhibited enhanced absorption and delayed elimination under DCM conditions, showing an opposite trend. These findings suggest that DCM may exert distinct regulatory effects on different bioactive ingredients of safflower, providing a comprehensive experimental basis for the clinical application of safflower-containing preparations.