Radiotherapeutic treatment consists of targeted application of radiation beams to a tumor but exposure of surrounding healthy tissue is inevitable. In the brain, ionizing radiation induces breakdown of the blood-brain barrier by effects on brain microvascular endothelial cells. Damage from directly irradiated cells can be transferred to surrounding non-exposed bystander cells, known as the radiation-induced bystander effect. We investigated involvement of connexin channels and paracrine signaling in radiation-induced bystander DNA damage in brain microvascular endothelial cells exposed to focused X-rays. Irradiation caused DNA damage in the directly exposed area, which propagated over several millimeters in the bystander area. DNA damage was significantly reduced by the connexin channel-targeting peptide Gap26 and the Cx43 hemichannel blocker TAT-Gap19. ATP release, dye uptake, and patch clamp experiments showed that hemichannels opened within 5 min post irradiation in both irradiated and bystander areas. Bystander signaling involved cellular Ca2+ dynamics and IP3, ATP, ROS, and NO signaling, with Ca2+, IP3, and ROS as crucial propagators of DNA damage. We conclude that bystander effects are communicated by a concerted cascade involving connexin channels, and IP3/Ca2+, ATP, ROS, and NO as major contributors of regenerative signal expansion.