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Over-representation analysis (ORA) is one of the commonest pathway analysis approaches used for the functional interpretation of metabolomics datasets. Despite the widespread use of ORA in metabolomics, the community lacks guidelines detailing its best-practice use. Many factors have a pronounced impact on the results, but to date their effects have received little systematic attention. Using five publicly available datasets, we demonstrated that changes in parameters such as the background set, differential metabolite selection methods, and pathway database used can result in profoundly different ORA results. The use of a non-assay-specific background set, for example, resulted in large numbers of false-positive pathways. Pathway database choice, evaluated using three of the most popular metabolic pathway databases (KEGG, Reactome, and BioCyc), led to vastly different results in both the number and function of significantly enriched pathways. Factors that are specific to metabolomics data, such as the reliability of compound identification and the chemical bias of different analytical platforms also impacted ORA results. Simulated metabolite misidentification rates as low as 4% resulted in both gain of false-positive pathways and loss of truly significant pathways across all datasets. Our results have several practical implications for ORA users, as well as those using alternative pathway analysis methods. We offer a set of recommendations for the use of ORA in metabolomics, alongside a set of minimal reporting guidelines, as a first step towards the standardisation of pathway analysis in metabolomics. Author summary: Metabolomics is a rapidly growing field of study involving the profiling of small molecules within an organism. It allows researchers to understand the effects of biological status (such as health or disease) on cellular biochemistry, and has wide-ranging applications, from biomarker discovery and personalised medicine in healthcare to crop protection and food security in agriculture. Pathway analysis helps to understand which biological pathways, representing collections of molecules performing a particular function, may be involved in response to a disease phenotype, or drug treatment, for example. Over-representation analysis (ORA) is perhaps the most common pathway analysis method used in the metabolomics community. However, ORA can give drastically different results depending on the input data and parameters used. Here, we have established the effects of these factors on ORA results using computational modifications applied to five real-world datasets. Based on our results, we offer the research community a set of best-practice recommendations applicable not only to ORA but also to other pathway analysis methods to help ensure the reliability and reproducibility of results. [ABSTRACT FROM AUTHOR]