Activated dendritic cells (DC) induce and polarize T-cell responses by expression of distinct maturation markers and cytokines. This study systematically investigated the capacity of different biotechnically relevant yeast species and strains including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Candida glabrata to initiate maturation of human DC. As important prerequisite for T-cell activation, all yeasts were shown to effectively induce, though to a different extent, the expression of the activation marker CD83, the co-stimulatory molecules CD80, CD86, CD54, CD58, and CD40, as well as the antigen-presenting molecules MHCs I and II. Furthermore, yeast-activated DC secreted various cytokines including inflammatory TNF-α, IL-6, IL-8, and IL-1β or T-cell polarizing IL-12, IL-10, IL-23, and IL-27. Variability was observed in the expression of TNF-α, IL-6, IL-8, IL-1β, and IL-10 in response to the tested yeasts, whereas expression levels of IL-12, IL-23, and IL-27 were similar. Interestingly, maturation marker expression and cytokine secretion were not negatively affected after application of yeast mutants with altered cell wall mannoprotein structure (Δmnn11) or defective in protein N-glycosylation (Δost3), indicating that elongated cell wall mannoproteins at the outer yeast cell surface are not a prerequisite for the observed yeast-mediated DC maturation. Thus, our data provide a valuable basic knowledge for the future design of effective yeast-based delivery approaches.