Presently, the study of influence of the quantity and location of weak interlayers on the stability of surrounding rock in horizontally-layered mudstone with weak interlayers remains insufficient. A case study is conducted on the Guandoushan single-track railway tunnel, and a three-dimensional numerical model of the tunnel with multiple weak interlayers is established using the three-dimensional distinct element code discrete element software. The displacement characteristics of the surrounding rock and the development of plastic zones are obtained, and the stability of the surrounding rock is analyzed. The results indicate the following: (1) The number of weak interlayers has a significant impact on the stability of tunnel surrounding rock. As the number of weak interlayers increases, the displacement of horizontally-layered surrounding rock gradually increases, and the area of plastic zones progressively expands. (2) The relative positional relationship between weak interlayers and the tunnel face is crucial for the stability of surrounding rock. When the number of weak interlayers is two (Case BC), the total displacement and plastic zone area of the surrounding rock are smaller than those in the case with one weak interlayer (Case A). Additionally, when weak gypsum layers are located in critical areas of the tunnel (e.g., the crown), their impact on structural stability becomes more pronounced, potentially leading to a significant decline in tunnel stability. (3) Variance analysis of the maximum total displacement of surrounding rock under different quantities and positions of weak gypsum layers is conducted using IBM SPSS software. The calculated P-values for the two factors are 0.037 and 0.019, respectively. Thus, both the quantity and location of weak gypsum layers have a statistically significant impact on structural stability at the 0.05 level, with the quantity of weak gypsum layers exhibiting greater parametric sensitivity than their location.