The quest for an efficacious HIV vaccine has resulted in several clinical trial failures, including the Step Trial, which used a replication-incompetent adenovirus (AdV) vector called human adenovirus type 5 (HAdV-5). Despite eliciting strong cellular immune responses, these trials were prematurely halted due to statistical futility resulting from increased HIV acquisition in vaccinated individuals. The Step Study showed increased HIV susceptibility in HAdV-5 baseline seropositive subjects, which complicates the use of HAdV-5 vectors since pre-existing neutralizing antibodies (nAb) to HAdV-5 are prevalent worldwide. Sampling the unique immunological phenotypes of the rectal mucosa - the site of HIV infection in the Step Study, and of AdV persistence and trafficking - could help explain this trial, since only peripheral blood (PBMC) was collected from subjects. We obtained rectal lamina propria T lymphocytes (rLPL) from a rhesus macaque (RM) model vaccinated with a species-specific simian Ad type 7 (SAdV-7) or HAdV-5 vector. We hypothesized that AdV-based vector vaccination in macaques previously exposed to AdV would activate AdV-specific CD4+ memory T cells that are either already harbored in the gastrointestinal mucosa or traffic to this original site of infection. The increased percentage of activated target cells upon SIV exposure would create a favorable environment for increased SIV susceptibility. We show an increase in both AdV-specific and activated CD4+ memory T cells in the rLPL after AdV vector vaccination relative to baseline, without significant changes in PBMCs. This led us to a proof-of-concept study where we collected PBMCs and rLPL from 10 SAdV-7 vector vaccinated and 10 placebo vaccinated RMs at baseline and week 2 post-vaccination. We performed low-dose escalating intra-rectal challenges weekly with SIVmac251 until SIV acquisition or 16 weeks post-vaccination. If we demonstrated increased SIV infection in the vaccine group, this would show evidence of activation-induced SIV susceptibility. Although we did not see an alteration in CD4+ memory T cell activation, we saw a trend towards SIV acquisition in the SAdV-7 vector vaccinated group versus placebo. Future studies should be cautious with the use of AdV vectors for mucosally transmitted pathogens, even for rare AdV serotypes due to T cell cross-reactivity and CD4+ memory T cell activation-induced susceptibility within mucosal sites.