Background/Objective: Vitamin D plays an important role in musculoskeletal health; however, its association with skeletal muscle microvascular perfusion has not been clearly defined using quantitative imaging techniques. To investigate the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and skeletal muscle microvascularity using an advanced ultrasound-based microvascular imaging method. Methods: In this cross-sectional study a total of 141 healthy adults were stratified into two groups according to serum 25(OH)D concentration (<20 ng/mL, n = 71; ≥20 ng/mL, n = 70). Ultra-Micro-Angiography was used to quantitatively assess the vascular index (VI), while cross-sectional area (CSA) measurements were obtained for the flexor carpi radialis, biceps brachii, and tibialis anterior muscles. Group comparisons and receiver operating characteristic (ROC) analyses were performed to evaluate the discriminative performance of microvascular parameters. Results: CSA values did not differ significantly between the groups. In contrast, VI values were significantly higher in participants with higher serum 25(OH)D levels across all examined muscles (p < 0.001). Among the evaluated parameters, the biceps brachii VI demonstrated excellent diagnostic performance in distinguishing between groups (AUC = 0.992; optimal cut-off = 1.25). Conclusions: Serum 25(OH)D levels are strongly associated with skeletal muscle microvascularity independent of muscle size. These findings demonstrate a strong association between serum 25-hydroxyvitamin D concentration and skeletal muscle microvascularity and highlight the potential of ultra-micro-angiography as a non-invasive imaging approach for detecting microvascular differences related to vitamin D status.