Objective To investigate the effect of prolonged stroboscopic illumination exposure on the growth of eyeball of guinea pig. Methods Thirty 2-week-old guinea pigs were randomized into three groups (n=10 for each). Two strobe-reared groups were raised with 20 Hz sinusoidal and 20 Hz square wave stroboscopic illumination, respectively. The control group received usual light illumination.The illumination intensity was 500 lux. All animals underwent refraction and biometric measurements prior to and after 2, 4, 6 and 8 weeks of treatment. Finally, flash electroretinograms were compared, and retinal microstructures were examined.Results There was a significant correlation between refractive errors and axial eye elongation, and myopia increasing was observed with eye elongation. After 8 weeks of treatment, the animals raised in 20 Hz sinusoidal and 20 Hz square wave stroboscopic illumination were (-0.75±0.79)D and (-1.50±0.91)D more myopic than the group raised in continuous illumination. The implicit time of the a-wave was delayed by 3.8 and 7.9 ms, respectively. No significant difference was found in retinal ultrastructures among the three groups.Conclusions Chronic exposure to 20 Hz sinusoidal or square wave stroboscopic illumination alters the emmetropization of the guinea pig eye to some extent.