Aims: To assess the safety and feasibility of acoustic radiation force impulse (ARFI) elastography in the eyes of rats. Material and methods: One hundred and twenty-six SD rats were divided into 7 groups. Group 1 was the control group and Group 2 to Group 7 were investigated by ARFI elastography using increased powers of 5%, 10%, 20%, 50%, and 100% and repetitions of 5 or 10. The changes of ocular architecture structures and functions were examined by ophthalmoscopic, histopathologic, and light reflex examination, and the expression level of IL-6, IL-8, and TNF-alpha was determined in days 1, 3, and 7 after ARFI elastography, respectively. Results: A satisfactory ARFI elastography was obtained in all SD rats. The shear wave velocity (SWV) values of soft tissues behind the eyeball were significantly greater than those of the eyes (1.89 +/- 0.95 vs. 0.84 +/- 0.36 m/s, p<0.05). The SWV values of eyes and the soft tissues behind the eyeball did not differ among any groups at any time-point (p>0.05). The SD rats of each group showed good light perception, pupillary light reflex, and avoidance reaction induced by the sudden bright light from the dark environment at any time. In each group, ocular architecture structures were well preserved, and with a normal expression level of IL-6, IL-8, and TNF-alpha (p>0.05). Conclusions: Diagnostic ARFI elastography is a safe and feasible ultrasonic imaging mode and may potentially be applied for human eyes in its present form.