Objective: Candida albicans is a critical priority fungal pathogen and one of the most common causes of invasive candidiasis in immunocompromised patients. The high toxicity of existing antifungals and the spread of drug-resistant fungi create a need for new agents, for which antimicrobial peptides (AMPs) with both antifungal and immunomodulatory effects are promising candidates. Methods: In this study, we used real-time PCR and sandwich ELISA to investigate the immunomodulatory effects on epithelial and immune cells of the tobacco defensin NaD1, which exhibits fungicidal activity against C. albicans, as well as its modified analogs NaD1-2, NaD1-3, and NaD1-4 with reduced cytotoxicity. Results and Discussion: We demonstrate that NaD1 exerts immunomodulatory effects on a Caco-2 cell monolayer modeling the human intestinal epithelial barrier, and its action differs from that of the human β-defensin HBD2. In the absence of infection, NaD1 at a concentration of 2 μM activates epithelial immune defense after 4 h of cell stimulation by upregulating the expression of genes encoding CXCL8, cathelicidin LL-37, and the patternrecognition receptors Dectin1 and TLR9, while not affecting IL-1β production. Under C. albicans infection, NaD1 predominantly exhibits antiinflammatory properties. In contrast, the effect of HBD2 becomes pronounced upon prolonged stimulation (24 h) and is proinflammatory both in the presence and absence of infection. Furthermore, unlike the tobacco defensin, the modified NaD1 analogs show virtually no immunomodulatory effects and do not affect IL-1β production by THP-1-derived macrophages. Finally, the TLR2 inhibitor C29 and the TLR4 inhibitor C34 reduce the stimulatory effect of NaD1 on IL-1β production by macrophages. Conclusions: Thus, the tobacco defensin NaD1, unlike its modified analogues, exhibits immunomodulatory activities on human epithelial and immune cells, and the TLR2 and TLR4 receptors may be involved in mediating these effects.