Objectives: Despite the widespread use of manganese (Mn) in industrial settings and its association with adverse neurological outcomes, a validated and reliable biomarker for Mn exposure is still elusive. Here, we utilize targeted metabolomics to investigate metabolic differences between Mn-exposed and -unexposed workers, which could inform a putative biomarker for Mn and lead to increased understanding of Mn toxicity.Methods: End of shift spot urine samples collected from Mn exposed (n = 17) and unexposed (n = 15) workers underwent a targeted assay of 362 metabolites using LC-MS/MS; 224 were quantified and retained for analysis. Differences in metabolite abundances between exposed and unexposed workers were tested with a Benjamini-Hochberg adjusted Wilcoxon Rank-Sum test. We explored perturbed pathways related to exposure using a pathway analysis.Results: Seven metabolites were significantly differentially abundant between exposed and unexposed workers (FDR ≤ 0.1), including n-isobutyrylglycine, cholic acid, anserine, beta-alanine, methionine, n-isovalerylglycine, and threonine. Three pathways were significantly perturbed in exposed workers and had an impact score >0.5: beta-alanine metabolism, histidine metabolism, and glycine, serine, and threonine metabolism.Conclusion: This is one of few studies utilizing targeted metabolomics to explore differences between Mn-exposed and -unexposed workers. Metabolite and pathway analysis showed amino acid metabolism was perturbed in these Mn-exposed workers. Amino acids have also been shown to be perturbed in other occupational cohorts exposed to Mn. Additional research is needed to characterize the biological importance of amino acids in the Mn exposure-disease continuum, and to determine how to appropriately utilize and interpret metabolomics data collected from occupational cohorts.