Connexins hemichannels (HCs) from adjacent cells form gap junctional channels that mediate cell-to-cell communication. Abnormal opening of "free" undocked HCs can produce cell damage and participate in the mechanism of disorders such as cardiac infarct, stroke, deafness, skin diseases, and cataracts. Therefore, inhibitors of connexin HCs have great pharmacological potential. Antibiotic aminoglycosides (AGs) have been recently identified as connexin HC inhibitors, but their antibiotic effect is an issue for the treatment of disorders where infections do not play a role. Herein, we synthesized and tested several amphiphilic AGs without antibiotic effect for their inhibition against connexin HCs, using a newly developed cell-based bacterial growth complementation assay. Several leads with superior potency than the parent compound, kanamycin A, were identified. Unlike traditional AGs, these amphiphilic AGs are not bactericidal and are not toxic to mammalian cells, making them better than traditional AGs as HC inhibitors for clinical use and other applications.