Ruxolitinib is a Janus kinase (JAK) 1/2 inhibitor, currently used in the treatment of myeloproliferative neoplasms. It exerts potent anti-inflammatory activity, but the involved molecular and cellular mechanisms remain poorly understood. In order to gain insights about this point, ruxolitinib effects towards expression of main inflammatory cytokines were studied in human macrophages, which constitute a key-cell type implicated in inflammation. Analysis of mRNA expression of cytokines (n = 84) by PCR array indicated that, among those induced by the pro-inflammatory stimulus lipopolysaccharide (LPS) (n = 44), 61.4% (n = 27) were repressed by 5 μM ruxolitinib. The major inflammatory cytokines, interleukin (IL) 6 and tumor necrosis factor α, were notably down-regulated by ruxolitinib at both the mRNA and protein level. Other repressed cytokines included IL27 and the chemokines CCL2, CXCL9, CXCL10 and CXCL11, but not IL1β. The interferon (IFN) β/JAK/signal transducer and activator of transcription (STAT) pathway, well-activated by LPS in human macrophages as demonstrated by increased secretion of IFNβ, STAT1 phosphorylation, and up-regulation of reference IFNβ-responsive genes, was concomitantly blocked by the JAK inhibitor. Most of cytokines targeted by ruxolitinib were shown to be regulated by IFNβ in a JAK-sensitive manner. In addition, counteracting the IFNβ/JAK/STAT cascade using a blocking monoclonal antibody directed against IFNβ receptor resulted in a similar profile of cytokine repression to that observed in response to the JAK inhibitor. Overall, these data provide evidence for ruxolitinib-mediated repression of inflammatory cytokines in human macrophages through inhibition of the LPS/IFNβ/JAK/STAT signalling pathway, which probably contributes to the anti-inflammatory effects of the JAK inhibitor.