The opportunistic fungal pathogen Trichosporon asahii (T. asahii) is an important causal agent of mortality in immunocompromised patients and associated with frequent relapses, even with sufficient antifungal treatment. Investigating the proteomes of initial and recurrent isolates may help to identify within-host adaptive changes. In this study, using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS) technology, we analyzed the proteomes of two T. asahii strains that were isolated 15 years apart from the same patient who suffered initial and recurrent episodes of systemic disseminated trichosporonosis. A total of 597 differentially expressed proteins were identified. Functional analysis showed that the increased proteins were primarily concentrated on peptide/protein/energy/drug metabolism and translation. Most of the results were determined to be consistent with the findings of phenotypic assays, such as tests for drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays. Moreover, we performed multiple reaction monitoring (MRM) mass spectrometry to verify 27 candidate proteins, and the results of this experiment were also highly consistent with the results of the TMT analysis. Therefore, to the best of our knowledge, these data provide the first molecular evidence of how the T. asahii proteome changes related to host-specific adaptation during human infection. Significance Systemic infection with Trichosporon asahii (T. asahii) has recently been recognized as an important causal agent of mortality in immunocompromised patients. Although triazole treatment usually works efficiently in the early phase of infection, many patients relapse. Hence, comparative analyses of the proteomics of initial and recurrent isolates may reveal evidence of adaptive changes within the host. Our study demonstrates that the recurrent strain has undergone proteomic changes using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS). Moreover, the results of phenotypic assays, including drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays, were highly consistent with the proteomic changes, and multiple reaction monitoring (MRM) verification also showed similar trends to the TMT results. In summary, our study is the first to investigate the adaptation of T. asahii under pressure from antifungal chemotherapy and host immune responses.