Background <p>Hematopoietic stem and progenitor cells (HSPCs) are crucial for blood production and regeneration. While their function is known to be regulated by diverse physical cues, the impact of pervasive radiofrequency electromagnetic fields (RF-EMF), particularly through non-thermal radiofrequency radiation (RFR) mechanisms, remains poorly understood.</p> Methods <p>We conducted colony-forming unit (CFU) assay in vitro and competitive transplantation assay in vivo to evaluate whether RFR influences hematopoiesis reconstitution capacity. Subsequently, the effects of RFR preconditioning on hematopoietic injury induced by ionizing radiation in mice were assessed by continuously monitoring the peripheral blood, HSPCs number, and colony-forming units. The influence of RFR on radioprotection unit frequency was evaluated using multiple gradients, non-competitive mouse transplantation models. Seahorse XF assays were employed to characterize cellular energy metabolic status, while specific fluorescent probes were utilized to detect calcium ion (Ca²⁺) levels in distinct cellular compartments using flow cytometry. Transcriptomic profiling was used to uncover the underlying mechanisms. HSPCs were pretreated with plasma membrane Ca²⁺-ATPase (PMCA) inhibitor prior to RFR exposure, and Seahorse assays along with CFU assay and competitive transplantation assay were performed to compare whether PMCA inhibition could abrogate RFR-induced HSPCs function change. To investigate the mechanism by which RFR enhanced PMCA activity inducing Ca²⁺ efflux, we performed fluorescence recovery after photobleaching (FRAP) assays to detect membrane fluidity.</p> Results <p>Non-thermal 2856&#xa0;MHz RFR enhanced HSPCs colony formation activity and reconstitution capacity, without compromising the multilineage differentiation homeostasis. RFR preconditioning accelerated hematopoietic recovery following ionizing radiation and increased radioprotection unit frequency. Mechanistically, RFR increased plasma membrane fluidity which potentiates PMCA activity, resulting in elevated Ca²⁺ efflux and reduced intracellular Ca²⁺ levels. These cellular alterations ultimately contributed to maintaining HSPCs in a low metabolic state, and consequently improving their functional capacity. Pharmacological inhibition of PMCA abolished both the functional enhancement and metabolic suppression.</p> Conclusion <p>Our results provided the first evidence that non-thermal RFR can improve HSPCs function. The central mechanism involved RFR-induced plasma membrane fluidity, activation of PMCA, thus accelerating Ca²⁺ efflux and maintaining HSPCs in a metabolically quiescent state. This work provided transformative insights into electromagnetic field biology and potential transplantation strategies for radiation-induced hematopoietic injury.</p>