The hippocampus plays a crucial role in brain function and is one of the important areas of concern in closed head injury. Hippocampal injury is related to a variety of factors including the strength of mechanical load, animal age, and helmet material. To investigate the order of these factors on hippocampal injury, a three-factor, three-level experimental protocol was established using the L9(34) orthogonal table. A closed head injury experiment regarding impact strength (0.3MPa, 0.5MPa, 0.7MPa), rat age (eight- week-old, ten-week-old, twelve-week-old), and helmet material (steel, plastic, rubber) were achieved by striking the rat's head with a pneumatic-driven impactor. The number of hippocampal CA3 cells was used as an evaluation indicator. The contribution of factors to the indicators and the confidence level were obtained by analysis of variance. The results showed that impact strength was the main factor affecting hippocampal injury (contribution of 89.2%, confidence level 0.01), rat age was a secondary factor (contribution of 8.9%, confidence level 0.05), and helmet material had no significant effect on hippocampal injury (contribution less than 1.9%). This paper provides a method to distinguish factors affecting hippocampal injury.