The mechanisms behind reported decreases in plasma insulin and glucagon during hemodialysis (HD) are not clear. Here, we investigated these mechanisms during HD treatment and the characteristics of insulin and glucagon removal when using two super high-flux membranes. In an experimental study, clearance, adsorption rates, and reduction rates of insulin and glucagon were investigated when using cellulose triacetate (CTA) and polysulfone (PS) membranes in a closed circuit using bovine blood. In a clinical study, 20 diabetes patients with end-stage kidney disease who were stable on HD were randomly selected for two HD sessions with two different membranes. At 1 h after the initiation of HD, insulin and glucagon clearance were measured, and the reduction rates were also investigated. In the experimental study, the PS membrane showed significantly higher clearance, adsorption rates, and reduction rates of insulin and glucagon compared with the CTA membrane. Although glucagon was detected in the ultrafiltration fluids in both membranes, insulin was absent in the PS membrane. In the clinical study, both membranes showed significant reductions in plasma insulin and glucagon at each time point. The PS membrane showed significantly higher insulin clearance and reduction rates compared with the CTA membrane. The two membranes showed no significant difference in glucagon clearance, but the glucagon reduction rate was significantly higher with the PS membrane. Our findings show that HD with the two super high-flux membranes used removes significant amounts of glucoregulatory peptide hormones from plasma in patients with diabetes and end-stage kidney disease, potentially affecting their glucose metabolism.