Spinach is an important crop, cultivated worldwide for its dark green leaves that are often consumed in salad mixes. Nearly 350,000 tons of spinach are produced in the U.S. every year and nearly 40 % of which are organically produced. Downy mildew of spinach, caused by the oomycete pathogen Peronospora effusa, remains a serious threat to production since synthetic fungicides cannot be applied for disease control in organic production. The use of resistant cultivars is an effective strategy to manage downy mildew, but much of the underlying genetic basis of susceptibility and resistance is not established in the spinach-downy mildew interaction. In this study, transcriptional profiles of P. effusa-inoculated resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, were monitored at an early stage of pathogen establishment (24 hours post inoculation, hpi) to a late stage of symptom expression and sporulation (168 hpi). Approximately 10,000 (24 hpi mock) to 12,000 (24 and 168 hpi) genes were differentially expressed. Major up-regulated genes linked to the host defense included those encoding a zinc finger CCCH protein, a 1-aminocyclopropane-1-carboxylate oxidase homolog, an LRR receptor-like serine/threonine-protein kinase, WRKY transcription factor, nucleoside diphosphate kinase, pectin methyltransferase QUA2, and pollen receptor-like kinase. This study provides preliminary information for elucidating molecular mechanisms of spinach- P. effusa interactions and candidate genes for resistance against infection.