Background <p>Relapsed/refractory (R/R) acute myeloid leukemia (AML) remains difficult to treat due to limited actionable targets and frequent drug resistance. Integrated analyses of multiple AML cohorts identified <i>S100A9</i> as a candidate factor associated with disease aggressiveness and suboptimal therapeutic response in R/R AML.</p> Methods <p>We combined genetic perturbation of S100A9 with mitochondrial Ca²⁺ measurements to define its functional role in AML cells. A structure-guided virtual screening strategy was then used to identify small molecules with direct affinity for S100A9, followed by biochemical validation and anti-leukemic profiling in AML cell lines, primary patient samples, normal hematopoietic cells, and xenograft models. Transcriptomic (RNA-seq) and protein assays were performed to characterize pathway changes induced by the lead compound.</p> Results <p>S100A9 modulation altered mitochondrial Ca²⁺ homeostasis and AML cell fitness. We identified B2, a novel S100A9-binding small molecule that reduces S100A9 abundance and is associated with increased mitochondrial Ca²⁺ accumulation. RNA-seq and immunoblotting demonstrated concomitant attenuation of survival signaling, including reduced STAT5 and AKT activation. B2 preferentially impaired S100A9-high AML cell lines and primary samples with minimal toxicity to normal hematopoietic cells, and significantly reduced leukemia burden in xenograft models.</p> Conclusions <p>These findings establish S100A9 as a regulator of mitochondrial Ca²⁺ homeostasis in AML and support B2 as a translational candidate that targets mitochondrial vulnerabilities and downstream survival pathways in R/R AML.</p>