Introduction MicroRNAs (miRNAs) play important roles in regulating biological procees at the posttranscriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expreion. Appropriate normalisation of RTqPCR data is important to ensure reliable results. The aim of the present study was to identify stably expreed miRNAs applicable as normaliser candidates in future studies of miRNA expreion in rectal cancer. Materials and Methods We performed high-throughput miRNA profiling (OpenArray1) on ten pairs of laser microdiected rectal cancer tiue and adjacent stroma. A global mean expreion normalisation strategy was applied to identify the most stably expreed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro diected rectal cancer tiue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tiue and normal rectal mucosa. Results From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expreed, both in malignant and stromal tiue. In addition, NormFinder confirmed high expreion stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expreion level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation). Conclusion We recommend the mean expreion of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expreion analyses by RT-qPCR on rectal cancer tiue.