<p>DOT1L, acting as a key epigenetic regulator, plays important roles in various biological processes, offering significant insights for the development of new cancer therapeutic strategies. This study investigates the impact of DOT1L on pyroptosis in osteosarcoma and its molecular mechanisms. Bioinformatics analysis was performed to identify genes associated with DOT1L. Western blotting was used to detect the expression levels of relevant proteins; flow cytometry was used to assess apoptosis in Saos-2 cells; transmission electron microscopy was used to observe the number of pyroptotic bodies in Saos-2 cells; subcutaneous xenograft experiments in nude mice were used to evaluate the progression of osteosarcoma; immunohistochemical staining was used to detect the expression of DOT1L, p-SYK, p-EGFR, p-SHP2, and NLRP3 in tumor tissues. Bioinformatics analysis revealed that DOT1L is associated with H3K79me2/3. Under hypoxic conditions and with cGAMP treatment, DOT1L promoted the expression of H3K79me2/3, SYK, EGFR, p-SYK, p-EGFR, p-STING, p-TBK1, p-IRF3, P53, NLRP3, IL-1β, GSDMD, and apoptosis in Saos-2 cells, while inhibiting the expression of p-SHP2 and SHP2. DOT1L mediated the SYK/EGFR/SHP2 signaling pathway to increase the number of pyroptotic bodies in Saos-2 cells. Additionally, DOT1L suppressed the progression of osteosarcoma and enhanced the expression of p-SYK, p-EGFR, and NLRP3 in tumor tissues, while inhibiting p-SHP2 expression. DOT1L suppressed the progression of osteosarcoma by modulating SYK/EGFR/P53 and SHP2-induced STING-NLRP3-pyroptosis signaling.</p>