Background <p>Venetoclax (VEN) in combination with hypomethylating agents has emerged as a pivotal therapy for elderly acute myeloid leukemia (AML) patients ineligible for intensive chemotherapy. However, monocytic AML exhibit greater resistance to VEN-based regimens compared to non-monocytic AML. Identifying exploitable vulnerabilities will mitigate resistance and relapse.</p> Methods <p>We conducted a comprehensive analysis of VEN resistance mechanisms in monocytic AML by integrating bulk AML datasets, single-cell RNA sequencing (scRNA-seq) of AML patient bone marrow and patient-derived xenograft (PDX) models, as well as lipidomic sequencing of induced VEN-resistant cell lines. Additionally, we examined the monocytic markers in VEN-resistant cell lines and assessed VEN sensitivity after knocking down the key sphingolipid metabolism gene <i>ASAH1</i>.</p> Results <p>Analysis of bulk RNA-seq data revealed elevated expression of sphingolipid metabolism genes in the French-American-British (FAB) M5 subtype, which exhibited poor response to VEN-based treatment. Further analysis of scRNA-seq data showed that monocytic AML cells surviving VEN treatment demonstrated the highest sphingolipid metabolism score, particularly in <i>CD14</i>⁺<i>ITGAX</i>⁺ monocytic AML cells. Notably, induced VEN-resistant cell lines exhibited significantly increased monocytic markers and differential sphingolipid metabolism profiles compared to parental cells. Among the key regulators of sphingolipid metabolism, <i>ASAH1</i> was upregulated, while SPHK1 was downregulated. Knocking down <i>ASAH1</i> enhanced VEN sensitivity without reducing the expression of monocytic markers CD14/CD64.</p> Conclusions <p>These findings suggest that aberrant sphingolipid metabolism contribute to AML resistance to VEN.</p>