Abstract Background Dupuytren’s disease is an inherited disorder in which patients develop fibrotic contractures of the hand. Current treatment strategies include surgical excision or enzymatic digestion of fibrotic tissue. MicroRNAs, which are key posttranscriptional regulators of genes expression, have been shown to play an important regulatory role in disorders of fibrosis. Therefore in this investigation, we apply high throughput next generation RNA sequencing strategies to characterize microRNA expression in diseased and healthy palmar fascia to elucidate molecular mechanisms responsible for pathogenic fibrosis. Methods We applied high throughput RNA sequencing techniques to quantify the expression of all known human microRNAs in Dupuytren’s and control palmar fascia. MicroRNAs that were differentially expressed between diseased and healthy tissue samples were used for computational target prediction using the bioinformatics tool ComiR. Molecular pathways that were predicted to be differentially expressed based on computational analysis were validated by performing RT-qPCR on RNA extracted from diseased and non-diseased palmar fascia biopsies. Results A comparison of microRNAs expressed in Dupuytren’s fascia and control fascia identified 74 microRNAs with a 2-fold enrichment in Dupuytren’s tissue, and 32 microRNAs with enrichment in control fascia. Computational target prediction for differentially expressed microRNAs indicated preferential targeting of collagens and extracellular matrix related proteins in control palmar fascia. RT-qPCR confirmed the decreased expression of microRNA targeted collagens in control palmar fascia tissues. Discussion Control palmar fascia show decreased expression of mRNAs encoding collagens that are preferentially targeted by microRNAs enriched in non-diseased fascia. Thus alterations in microRNA regulatory networks may play an important role in driving the pathogenic fibrosis seen in Dupuytren’s disease via direct regulatory effects on extracellular matrix protein synthesis. Conclusion Dupuytren’s fascia and healthy palmar fascia can be distinguished by unique microRNA profiles, which are predicted to preferentially target collagens and other extracellular matrix proteins.