Unfractionated heparin (UFH), a naturally occurring anionic polysaccharide, is widely used as an anticoagulant agent in clinical practice. When overdosed or used in sensitive patients, UFH may cause various risks and a UFH neutralizer needs to be administered immediately to reverse heparinization. However, the most common UFH neutralizer, protamine sulfate, often causes various adverse effects, some of which are life-threatening. Herein, we designed a highly biocompatible, oligoethylene glycol functionalized guanidinocalixarene (GC4AOEG) as an antidote against UFH. GC4AOEG and UFH exhibited a strong binding affinity, ensuring specific recognition and neutralization of UFH by GC4AOEGin vitroandin vivo. As a consequence, UFH-induced excessive bleeding was significantly alleviated by GC4AOEG in different mouse bleeding models. Additionally, no adverse effects were observed during these treatmentsin vivo. Taken together, GC4AOEG, as a strategically designed, biocompatible artificial receptor with strong recognition affinity towards UFH, may have significant clinical potential as an alternative UFH reversal agent.