Negative pressure wound therapy (NPWT) has significantly advanced wound care and continues to find new applications. Its effects at a molecular level however, remain a subject of debate. The aim of this systematic review is to summarize the current evidence regarding the molecular mechanisms of action of NPWT. Medline, Embase, EBSCO databases and clinical trial registries were searched from inception to January 2023. Clinical studies, animal models or in-vitro studies that quantitatively or semi-quantitatively evaluated the influence of NPWT on growth factors, cytokine or gene-expression in the circulation or wound-bed were included. Risk of Bias assessment was performed using the RoBANS tool for non-randomized studies, the COCHRANE's Risk of Bias 2(ROB-2) tool for randomized clinical studies, OHAT tool for in-vitro studies or the SYRCLE tool for animal model studies. A descriptive summary was collated and the aggregated data is presented as a narrative synthesis. This review included 19 clinical studies, 11 animal studies and 3 in-vitro studies. The effects of NPWT on 43 biomarkers and 17 gene expressions were studied across included studies. NPWT stimulates modulation of numerous local and circulating cytokines and growth factor expressions to promote an anti-inflammatory profile. This is most likely achieved by downregulation of TNFα, upregulation of VEGF, TGF-β and fibronectin.