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Simple Summary: This study addresses the limited understanding of the molecular consequences of low-dose ionizing radiation (LDIR), a relatively unexplored area compared to high-dose radiation methods. The research focuses on profiling RNA species crosslinked to the RNA-binding protein human antigen R (HuR) using LDIR and high throughput RNA sequencing. The developed method involves isolating RNA fragments crosslinked to HuR through LDIR and immunoprecipitation followed by RNA sequencing, revealing rapid degradation of target mRNAs (e.g., PAX6, ZFP91, NR2F6, and CAND2) in human cell lines. Notably, the downregulation of PAX6 and NR2F6 is identified as a protein mediating the beneficial effects of LDIR on cell viability. The findings underscore the significance of investigating post-transcriptional gene regulation under LDIR conditions. Overall, this research introduces a valuable approach for understanding the impact of LDIR on RNA dynamics, shedding light on the molecular consequences and potential therapeutic applications of low-dose ionizing radiation. Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide–protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR. [ABSTRACT FROM AUTHOR]