Objective: Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MB _cAbVcam1-5 ) and microbubble with a control nanobody (MB _VHH2E7 ) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MB _cAbVcam1-5 and MB _VHH2E7 . Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×10 ⁵ per microbubble. Compared with MB _VHH2E7 , MB _cAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm ² while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MB _cAbVcam1-5 in early ( P =0.0003 versus MB _VHH2E7 ) and late atherosclerosis ( P =0.007 versus MB _VHH2E7 ); in wild-type mice, there were no differences between MB _cAbVcam1-5 and MB _VHH2E7 . CEUMI in human endarterectomy specimens showed a 100% increase in signal for MB _cAbVcam1-5 versus MB _VHH2E7 (20.6±27.7 versus 9.6±14.7, P =0.0156). Conclusions: CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.