Cellular immunotherapy remains hindered in the context of solid tumors due to the immunosuppressive microenvironment, in which key endothelial cell adhesion molecules (CAM) are suppressed. Microbubble-mediated focused ultrasound is being explored for targeted immunotherapy and can exert local shear stress upon neighboring endothelial cells. However, fluid and microbubble-induced shear modulation of endothelial immunobiology is not well understood. Herein, the influence of both types of shear stress on human endothelial vein (HUVEC) and brain endothelial (HBEC-5i) CAM expression and secretion of over 90 cytokines using acoustically coupled microscopy is examined. Fluid flow results in time-dependent modulation of CAM expression, where ICAM-1 peaked at 4 h (1.98-fold,  < 0.001, HUVEC) and 24 h (1.56-fold,  < 0.001, HBEC-5i). While some chemokines are significantly enhanced (up to 16.2-fold;  < 0.001) from both endothelial cell types (e.g., IL-8, MCP-1, MCP-3), others are differentially expressed (e.g., CCL5, CXCL-16, SDF-1). Under ultrasound, ICAM-1 expression at 4 h increased (≈1.4-fold,  < 0.01) and resulted in significant large-magnitude ( < 0.05) differential expression of 20 cytokines, most of which have immune-activating function and within a subset of those induced by shear-flow. Microbubble-mediated ultrasound regulates ICAM-1 expression and the human endothelial secretome toward an immune cell recruitment paradigm, and thus may reinforce solid tumor cellular immunotherapy efforts.