Non-celiac wheat sensitivity (NCWS) is a recently recognized syndrome triggered by a gluten-containing diet. The pathophysiological mechanisms engaged in NCWS are poorly understood and, in the absence of laboratory markers, the diagnosis relies only on a double-blind protocol of symptoms evaluation during a gluten challenge. We aimed to shed light on the molecular mechanisms governing this disorder and identify biomarkers helpful to the diagnosis. By a genome-wide transcriptomic analysis, we investigated gene expression profiles of the intestinal mucosa of 12 NCWS patients, as well as 7 controls. We identified 300 RNA transcripts whose expression differed between NCWS patients and controls. Only 37% of these transcripts were protein-coding RNA, whereas the remaining were non-coding RNA. Principal component analysis (PCA) and receiver operating characteristic curves showed that these microarray data are potentially useful to set apart NCWS from controls. Literature and network analyses indicated a possible implication/dysregulation of innate immune response, hedgehog pathway, and circadian rhythm in NCWS. This exploratory study indicates that NCWS can be genetically defined and gene expression profiling might be a suitable tool to support the diagnosis. The dysregulated genes suggest that NCWS may result from a deranged immune response. Furthermore, non-coding RNA might play an important role in the pathogenesis of NCWS.