•Chemical composition of fermented bee pollen was globally analyzed.•Fermentation led to 668 differential metabolites in bee pollen.•Fermentation significantly changed the primary and secondary metabolites.•Fermentation transformed polyphenol glycosides to aglycones.•Fermentation increased the types and contents of phenolamides. Microbial fermentation can break the bee pollen wall. However, the global profiling of bee pollen metabolites under fermentation remains unclear. This study aims to comprehensively elucidate the changes in the composition of bee pollen after microbial fermentation. Ultra-performance liquid chromatography-electron spray ionization-mass spectrometry (UPLC-ESI-MS) based on widely targeted metabolomics analysis was used to compare the chemical composition of unfermented bee pollen (UBP) and fermented bee pollen (FBP). Among the 890 metabolites detected, a total of 668 differential metabolites (classified into 17 categories) were identified between UBP and FBP. Fermentation significantly increased the contents of primary metabolites such as 74 amino acids and derivatives, 42 polyunsaturated fatty acids and 66 organic acids, as well as some secondary metabolites such as 38 phenolic acids, 80 flavone aglycones and 22 phenolamides. The results indicate that fermentation is a promising strategy to improve the nutritional value of bee pollen.