The poultry red mite, Dermanyssus gallinae, is a haematophagous ectoparasite that represents a major challenge to the global egg-laying industry, affecting both poultry welfare and productivity. Control of D. gallinae infestations remains difficult and increasing concerns regarding resistance and regulatory restrictions highlight the need for novel, targeted and sustainable control strategies. As an obligate blood feeder, efficient processing of the blood meal is essential for nutrient acquisition and reproduction. Investigating the molecular processes underlying blood meal processing may therefore help identify potential vulnerabilities in mite physiology. The aim of this study was to use RNA interference (RNAi) to investigate genes involved in blood meal processing in D. gallinae. Target-specific double-stranded RNA (dsRNA) molecules were delivered to D. gallinae via an in vitro blood feeding system. Following dsRNA exposure, gene expression levels were assessed using real time quantitative PCR to evaluate the efficiency of RNAi-mediated gene knockdown. In addition, mites were monitored following feeding for changes in survival, egg production and indicators of blood meal processing, to assess potential physiological effects associated with gene silencing. To provide broader context for these findings, the effects of pathway disruption using chemical inhibition were also examined. Oral delivery of dsRNA through the blood meal resulted in knockdown of selected target genes associated with blood meal processing pathways in D. gallinae. RNAi-mediated silencing enabled investigation of downstream physiological processes following feeding. While gene knockdown resulted in measurable effects on mite fitness, these were more limited than those observed following chemical inhibition of the same pathway. Taken together, these findings suggest that RNAi may not always reflect the full effects of disrupting an entire biological pathway, but it remains a valuable tool for investigating gene function in D. gallinae and improving our understanding of the molecular processes underlying blood meal processing.