Simple SummaryMicrobeams are useful tools in studies on non-target effects, such as the radiation-induced bystander effect, and responses related to cytoplasmic irradiation. A micrometer or even sub-micrometer-level beam size enables the precise delivery of radiation energy to a specific target. Here we summarize the observations of the bystander effect and the cytoplasmic irradiation-related effect using different kinds of microbeam irradiators as well as discuss the cellular and molecular mechanisms that are involved in these responses. Non-target effects may increase the detrimental effect caused by radiation, so a more comprehensive knowledge of the process will enable better evaluation of the damage resulting from irradiation.Although direct damage to nuclear DNA is considered as the major contributing event that leads to radiation-induced effects, accumulating evidence in the past two decades has shown that non-target events, in which cells are not directly irradiated but receive signals from the irradiated cells, or cells irradiated at extranuclear targets, may also contribute to the biological consequences of exposure to ionizing radiation. With a beam diameter at the micrometer or sub-micrometer level, microbeams can precisely deliver radiation, without damaging the surrounding area, or deposit the radiation energy at specific sub-cellular locations within a cell. Such unique features cannot be achieved by other kinds of radiation settings, hence making a microbeam irradiator useful in studies of a radiation-induced bystander effect (RIBE) and cytoplasmic irradiation. Here, studies on RIBE and different responses to cytoplasmic irradiation using microbeams are summarized. Possible mechanisms related to the bystander effect, which include gap-junction intercellular communications and soluble signal molecules as well as factors involved in cytoplasmic irradiation-induced events, are also discussed.