The Macrophage-Inducible C-type Lectin receptor (Mincle) plays a critical role in innate immune recognition and pathology, and therefore represents a promising target for vaccine adjuvants. Innovative trehalose-based Mincle agonists with improved pharmacology and potency may prove useful in the development of Th17-mediated adaptive immune responses. Herein, we report on in vitro and in silico investigations of specific Mincle ligand-receptor interactions required for the effective receptor engagement and activation of Th17-polarizing cytokines. Specifically, we employed a library of trehalose benzoate scaffolds, varying the degree of aryl lipidation and regiochemistry that produce inflammatory cytokines in a Mincle-dependent fashion. In vitro interleukin-6 (IL-6) cytokine production by human peripheral blood mononuclear cells (hPBMCs) indicated that the lipid regiochemistry is key to potency and maximum cytokine output, with the tri-substituted compounds inducing higher levels of IL-6 in hPBMCs than the di-substituted derivatives. Additionally, IL-6 production trended higher after stimulation with compounds that contained lipids ranging from five to eight carbons long, compared to shorter (below five) or longer (above eight) carbon chains, across all the substitution patterns. An analysis of the additional cytokines produced by hPBMCs revealed that compound , tri-substituted and five carbons long, induced significantly greater levels of interleukin-1β (IL-1β), tumor necrosis factor- α (TNF-α), interleukin-23 (IL-23), and interferon- γ (IFN-γ) than the other compounds tested in this study. An in silico assessment of highlighted the capability of this analogue to bind to the human Mincle carbohydrate recognition domain (CRD) efficiently. Together, these data highlight important structure-activity findings regarding Mincle-specific cytokine induction, generating a lead adjuvant candidate for future formulations and immunological evaluations.