Lymph node (LN) stromal cells are critical regulators of immune reactions, yet their responses to different SARS-CoV-2 vaccines remain unexplored. Here, we immunized mice with clinically approved gene- and protein-based COVID-19 vaccines targeting viral spike (S) protein and analyzed the draining LN stroma using multimodal bioimaging, single-cell transcriptomics, and functional studies. We found that messenger RNA and adenovirus vector vaccines transfected lymphatic endothelial cell and fibroblastic reticular cell subsets in vivo and led to early local S protein production in the draining LN in a vaccine-specific manner. The vaccines induced rapid transcriptomic reprogramming of the LN stromal cells, which functionally altered scavenging and parenchymal transfer of lymph-borne antigens, formation of chemokine gradients, and migration of eosinophils within LNs. Thus, distinct vaccine formulations targeting S protein differentially prime the draining LN stromal cells before the arrival of migratory dendritic cells bearing immunogens.