Abstract Motivation: Many studies have investigated the differential expression of microRNAs (miRNAs) in disease states and between different treatments, tissues and developmental stages. Given a list of perturbed miRNAs, it is common to predict the shared pathways on which they act. The standard test for functional enrichment typically yields dozens of significantly enriched functional categories, many of which appear frequently in the analysis of apparently unrelated diseases and conditions. Results: We show that the most commonly used functional enrichment test is inappropriate for the analysis of sets of genes targeted by miRNAs. The hypergeometric distribution used by the standard method consistently results in significant P-values for functional enrichment for targets of randomly selected miRNAs, reflecting an underlying bias in the predicted gene targets of miRNAs as a whole. We developed an algorithm to measure enrichment using an empirical sampling approach, and applied this in a reanalysis of the gene ontology classes of targets of miRNA lists from 44 published studies. The vast majority of the miRNA target sets were not significantly enriched in any functional category after correction for bias. We therefore argue against continued use of the standard functional enrichment method for miRNA targets. Availability and implementation: A Python script implementing the empirical algorithm is freely available at http://sgjlab.org/empirical-go/. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.