Aging is associated with alterations in immune cell function, contributing to age-related diseases and frailty. As peripheral blood mononuclear cells (PBMCs) are key drivers of the immune response, we investigated their transcriptome using RNA-sequencing before and immediately after a single bout of high-intensity knee-extension exercise in young (young; = 7, 23 ± 4 yr) and older individuals (old; = 8, 65 ± 7 yr). We used bioinformatics analyses to identify the biological processes and pathways that may be altered with age and in response to acute exercise. At baseline, 665 genes differed between young and old, with notable differences in pathways involved in DNA damage/telomere stress-induced senescence, NAD signaling pathway, and oxidative stress-induced senescence. After the exercise bout, 53 genes were differentially expressed in young, whereas 1,026 genes changed in old. In young, the enriched processes and predicted pathways were linked to natural killer cells, whereas in old, these pathways were associated with cell signaling immune responses. Finally, 26 genes exhibited similar responses to exercise between groups, enriching the biological process of natural killer cell-mediated immunity regulation. Our findings indicate that PBMC gene expression and the response to acute exercise are altered with aging, where exercise induces more pronounced PBMC transcriptomic adaptations in the old. In addition, although aging is associated with increased expression of genes linked to cellular dysfunction and suppressed immune function, acute exercise attenuated these age-related differences by downregulating the genes related to those pathways. Finally, acute exercise activated similar immune-related pathways in both age groups. This study demonstrates that aging alters the transcriptional landscape of PBMCs at rest and in response to acute high-intensity exercise. Older adults exhibited greater transcriptomic responsiveness to exercise, particularly in pathways related to immune signaling and cellular stress. Notably, exercise elicited shared activation of NK cell-mediated processes across age groups, suggesting a conserved immunomodulatory effect. These findings provide molecular insight into how aging and exercise interact to shape immune cell function.