Interferon-alpha (IFN alpha) has one of the longest histories of use amongst cytokines in clinical oncology and has been applied for the treatment of many types of cancers. Due to its immune-activating properties, IFN alpha is also an attractive candidate for combinatory anti-cancer therapies. Despite its extensive use in animal tumor models as well as in several clinical trials, the different mechanisms underlying patient responses and affecting desirable clinical benefits are still under investigation. Here we show that in addition to its immune-activating properties, IFN alpha induces the expression of a key negative regulator, immunosuppressive PD-L1 molecule, in the majority of the specific immune cell populations, particularly in the dendritic cells (DC). DC can modulate immune responses by a variety of mechanisms, including expression of T-cell regulatory molecules and cytokines. Our results showed that treatment of DC with IFN alpha-2b led to pronounced up-regulation of surface expression of PD-L1 molecules, increased IL-6 and decreased IL-12 production. Moreover, we present evidence that IFN alpha-treated DC exhibited a reduced capacity to stimulate interferon-gamma production in T cells compared to control DC. This T-cell response after treatment of DC with IFN alpha was recovered by a pre-treatment with an anti-PD-L1 blocking antibody. Further analyses revealed that IFN a regulated PD-L1 expression through the STAT3 and p38 signaling pathways, since blocking of STAT3 and p38 activation with specific inhibitors prevented PD-L1 up-regulation. Our findings underline the important roles of p38 and STAT3 in the regulation of PD-L1 expression and prove that IFN alpha induces STAT3/p38-mediated expression of PD-L1 and thereby a reduced stimulatory ability of DC. The augmentation of PD-L1 expression in immune cells through IFN alpha treatment should be considered by use of IFN alpha in an anti-cancer therapy.