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.