MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate gene expression at the post‑transcriptional level. It has been demonstrated that miRNAs serve a crucial role in tissue development and the pathogenesis of numerous diseases. The aim of the current study was to investigate the alterations in miRNA expression in a cultured retinal ganglion cell line (RGC‑5 cells) following ionizing radiation injury. Cultured RGC‑5 cells were exposed to X‑rays at doses of 2, 4, 6 and 8 Gy using a medical linear accelerator. Alterations in cellular morphology were observed under a phase contrast microscope and cell viability was measured using the MTT assay. Subsequent to exposure to X‑ray radiation for 5 days, the viability of RGC‑5 cells was significantly reduced in the 6 and 8 Gy groups, accompanied by morphological alterations. Total RNA was then extracted from RGC‑5 cells and subjected to miRNA microarray analysis subsequent to exposure to 6 Gy X‑ray radiation for 5 days. The results of the microarray analysis indicated that the expression levels of 12 miRNAs were significantly different between the 6 Gy and control groups, including 6 upregulated miRNAs and 6 downregulated miRNAs. To verify microarray results, a reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis was performed. The data obtained from RT‑qPCR analysis was similar to that of the the microarray analysis for alterations in the expression of the 12 miRNAs. The results of the current study indicated that miRNA expression was sensitive to ionizing radiation, which may serve an important role in mechanisms of radiation injury in retinal ganglion cells.