Polymorphonuclear cells (PMN) are widely recognized as sophisticated killers during microbial infections. In recent years, PMN have been shown to interact and functionally interfere with other cells of the immune system. In this study, we investigated PMN-T cell interactions in an in vitro co-culture system. A relative increase in T cells in the co-culture was associated with the upregulation of CD66b expression on PMN. In addition, PMN were found to dose-dependently impair anti-CD3 induced CD4⁺ T cell activation, proliferation and viability. In a transwell co-culture system, proliferation of T cells was, however, enhanced which illustrates that suppression was contact-dependent. The addition of an arginase-inhibitor or blocking antibodies against calprotectin, but not myeloperoxidase (MPO), partially restored T cell proliferation. Furthermore, the presence of PMN in the co-culture dose-dependently increased the fraction of IFN-γ and IL-17 producing T cells and decreased the percentage of IL-10 producing CD4⁺ T cells. Altogether, these data show that there is cross-talk between PMN and T cells which, in non-inflammatory conditions, results in the effects described above. Further studies should investigate PMN-T cell functional interference in inflammatory situations and clarify the importance of this PMN-T cell cross-talk in the regulation of the immune response.