Aluminum (Al) toxicity is one of the most important limiting factors for crop yield in acidic soils. Bound Al gets converted into a toxic ionic state (Al3+) in acidic soil. Recent studies have shown that Al can act on the cell walls, cell membranes, organelles, and nuclei of microorganisms and affect substance and energy metabolism. To explore the gene expression at the transcriptional level under Al stress, we sequenced the transcriptome of Cryptococcus humicola, which is a highly Al-resistant yeast strain isolated from acidic soil and tolerates up to 200 mM Al3+. The screening conditions for genes from the control and experimental group were a false discovery rate (FDR) <0.05 and log 2|FC| > 1. A total of 4760 genes were differentially expressed, among which 3066 were upregulated and 1694 were downregulated. These genes control glycometabolism, protein synthesis, lipid metabolism and signalling pathways. Eleven selected differentially expressed genes were further validated using qRT-PCR. The results suggested that Al stress leads to complex responses in C. humicola. The effects of Al on the β-d-glucan and mannose contents and Al accumulation in the cell wall were determined. With an increase in the Al treatment time and concentration, the contents of β-d-glucan and mannose showed a trend of first increasing and then decreasing. Under Al treatment, the Al content of the cell wall also showed a trend of first increasing and then decreasing. These results suggested that Al accumulates in the cell wall and the cell wall plays a vital role in the Al resistance of C. humicola. The differentially expressed genes provide a foundation for the further study of Al tolerance in C. humicola.