Updated project metadata. Light plays an important role in the growth and differentiation of Lentinula edodes mycelia, and mycelial morphology is influenced by light wavelengths. The blue light-induced formation of brown film on the vegetative mycelial tissues of L. edodes is an important process. However, the mechanisms of L. edodes’ brown film formation, as induced by blue light, are still unclear. Using a high-resolution liquid chromatography-tandem mass spectrometry integrated with a highly sensitive immune-affinity antibody method, phosphoproteomes of L. edodes mycelia under red- and blue-light conditions were analyzed. A total of 11,224 phosphorylation sites were identified on 2,786 proteins, of which 9,243 sites on 2,579 proteins contained quantitative information. In total, 475 sites were up-regulated and 349 sites were down-regulated in the blue vs red group. To characterize the differentially phosphorylated proteins, systematic bioinformatics analyses, including gene ontology annotations, domain annotations, subcellular localizations, and Kyoto Encyclopedia of Genes and Genomes pathway annotations, were performed. These differentially phosphorylated proteins were correlated with light signal transduction, cell wall degradation, and melanogenesis, suggesting that these processes are involved in the formation of the brown film. Our study provides new insights into the molecular mechanisms of the blue light-induced brown film formation at the post-translational modification level.