Background <p>Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with few therapeutic options. Observational data suggest that type 2 diabetes mellitus (T2DM) might protect against ALS, yet the mechanisms are unclear. Clarifying whether glucose or lipid metabolism underpins this protective effect could guide targeted interventions.</p> Objective <p>This study aims to investigate if T2DM reduces ALS risk through glycemic or lipid pathways using a two-step Mendelian Randomization (MR) approach.</p> Methods <p>Summary-level genetic data were sourced from FinnGen (<i>n</i> = 440,735), MAGIC (<i>n</i> = 200,622), UK Biobank (<i>n</i> = 115,078), and Project MinE (<i>n</i> = 138,086). Two-sample MR assessed T2DM’s causal effect on ALS, followed by multivariable MR adjusting for glycemic traits to identify metabolic pathways. A two-step MR analyzed significant blood metabolites contributing to the T2DM-ALS relationship. Sensitivity analyses confirmed the robustness of these findings.</p> Results <p>T2DM exhibited a protective causal association with ALS (inverse variance weighting OR = 0.956, 95% CI 0.916–0.997, <i>p</i> = 0.037). Glycemic traits did not mediate this protection; instead, lipid metabolism played a role. Specifically, a 1 SD reduction in LDL diameter was linked to a 16.7% decrease in ALS risk, accounting for 24.4% of T2DM’s protective effect. Similarly, a 1 SD decrease in total esterified cholesterol (TEC) reduced ALS risk by about 13.2%, contributing to 13.3% of T2DM’s overall protective impact. No evidence of horizontal pleiotropy was observed.</p> Conclusion <p>T2DM’s protective influence on ALS primarily involves lipid rather than glucose pathways, highlighting TEC and LDL particle diameter as crucial mediators. Targeting lipid metabolism may offer new therapeutic strategies to reduce ALS risk or progression, potentially leading to focused nutritional interventions and biomarker development.</p>