Background Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the uncontrolled proliferation of immature myeloid cells. The proteasome subunit PSMD3, part of the 26S regulatory complex, has emerged as a potential oncogenic driver, but its role in AML still remains underexplored. Methods This study investigates the consequences of shRNA-mediated PSMD3 knockdown in three AML cell lines, MOLM-13, MOLM-14, and MV4-11, using RNA sequencing to better understand the role of PSMD3 in AML. Results The analysis of gene expression following PSMD3 silencing revealed both cell line-specific and shared pathway changes. In MV4-11, there was a downregulation of pathways associated with “Adaptive immune response”, “Phosphorylation”, “Regulation of retinoic acid receptor signaling pathway”, and “Regulation of MAPK cascade”. Pathways that were upregulated were associated with inflammation, such as “Positive regulation of cytokine production” and “Response to bacterium”. In MOLM-14, pathways that were downregulated were related to the cell cycle, such as “Cell cycle”, “Phosphorylation”, “Metabolism of vitamins and cofactors”, “VEGFA VEGFR2 signaling”, and “WNT signaling pathway”. Pathways that were upregulated were related to inflammation and included pathways such as “Neutrophil Degranulation”, “Immune Responses”, “Cytokine signaling in the immune system”, and “Phosphorylation”. In MOLM-13, the pathways that were downregulated were related to “Positive regulation of angiogenesis” and “Regulation of WNT signaling pathway”. The pathways that were upregulated were related to inflammation, such as “Neutrophil Degranulation”, “Inflammatory Responses”, “Interleukin-4 and Interleukin-13 signaling”, and Vitamin pathways. Conclusion This study has highlighted critical pathways involved in AML progression that are regulated by the 19S proteasome, particularly the roles of inflammation, immune response, and cell cycle regulation. Our findings indicate that AML cells leverage inflammatory and immune signaling pathways to create a pro-tumorigenic environment conducive to proliferation, while simultaneously downregulating cell cycle checkpoints to evade normal regulatory mechanisms. The unexpected upregulation of vitamin pathways, especially vitamins C and D, suggests that AML cells may use metabolic responses as a potential adaptive mechanism under therapeutic pressure. The implications of these findings are twofold. The upregulation of inflammatory pathways reinforces PSMD3’s role in maintaining a pro-tumorigenic inflammatory environment, through proteasome-regulated signaling to promote AML proliferation and survival. Targeting these pathways, through cytokine or TLR signaling inhibitors, or even targeting the proteasome itself, could be a promising therapeutic approach. The unexpected upregulation of vitamin pathways presents a therapeutic angle, as promoting differentiation in AML cells could reduce their malignancy and responsiveness to proliferative cues. Thus, these results suggest that a combination of anti-inflammatory and differentiation-inducing treatments might synergize with AML therapy in the context of 19S proteasome inhibition.