Tacrolimus is a widely used immunosuppressive agent. Although T cells are the main targets of these pharmacological drugs, antigen presentation may also be affected. Among antigen-presenting cells, plasmacytoid dendritic cells (PDCs) are the main source of type I interferons upon microbial challenge, and are involved in several diseases and autoimmune disorders. The aim of this study was to evaluate whether tacrolimus can modulate the function of PDCs in vitro. Maturation and function of PDCs was determined using flow cytometry, enzyme-linked immunosorbent assay and cytometry bead arrays. The effect of tacrolimus on PDCs was observed mainly when the cells were pretreated with the immunosuppressive agent before activation. Upon dinucleotide-oligodeoxynucleotide (CpG-ODN) activation, tacrolimus pretreated PDCs showed a significant reduction in the surface expression of co-stimulatory molecules and human leucocyte antigen D-related (HLA-DR) and secreted reduced levels of tumour necrosis factor (TNF)-alpha. These results show that tacrolimus treatment of PDCs impairs CpG-induced activation, which could affect the outcome of the immune response.