T follicular helper (T FH ) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident T FH cells have been fully characterized in human immunodeficiency virus (HIV) infection. However, the role of GC T FH cells in GC B cell responses following various simian immunodeficiency virus (SIV) vaccine regimens in rhesus macaques (RMs) has not been fully investigated. We characterized GC T FH cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants and systemically boosted with ALVAC-SIV M766 Gag/Pro/gp120-TM and SIV M766&CG7V gD-gp120 proteins formulated in alum hydroxide (ALVAC/Env) or DNA encoding SIVenv/ SIVGag/rhesus interleukin 12 (IL-12) plus SIV M766&CG7V gD-gp120 proteins formulated in alum phosphate (DNA&Env). Lymph nodes were biopsied in macaque subgroups prevaccination and at day 3, 7, or 14 after the 2nd Ad5hr-SIV prime and the 2nd vector/Env boost. Evaluations of GC T FH and GC B cell dynamics including correlation analyses supported a significant role for early GC T FH cells in providing B cell help during initial phases of GC formation. GC T FH responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia postinfection. Our results highlight the importance of early GC T FH cell responses for robust GC maturation and generation of long-lasting SIV-specific humoral responses at mucosal and systemic sites. Further investigation of GC T FH cell dynamics should facilitate development of an efficacious HIV vaccine.