As the first responder to immunological challenges, the innate immune system shapes and regulates the ensuing adaptive immune response. Many clinical studies evaluating the role of innate immunity in initiating vaccine-elicited adaptive immune responses have largely been confined to blood due to the inherent difficulty in acquiring tissue samples. However, the absence of vaccine-site and draining lymph node information limits the understanding of early events induced by vaccination that could potentially shape vaccine-elicited immunity. We, therefore, utilized a mouse model to investigate the spatiotemporal evolution of the immune response within the first 24 hours following intramuscular adenovirus serotype 26 (Ad26) vector vaccination in tissues. We show that the Ad26 vaccine-elicited innate immune response commences by 1 hour and rapidly evolves in tissues and blood within the first 24 hours, as reflected by the detection of cytokines, chemokines, cellular responses, and transcriptomic pathways. Furthermore, serum levels of IL-6, MIG, MIP-1α, MIP-1β, and TNF-α at 6 hours post-vaccination correlated with the frequency of vaccine-elicited memory CD8