In the French conventional railway track, an interlayer was created naturally through the interpenetration of ballast and subgrade under the effect of long-term train loading. Field investigation showed that the proportion of ballast grains decreased over depth in the interlayer. Moreover, the water content of interlayer soils varied depending on the weather conditions, which can strongly affect the mechanical behavior of interlayer soil. In this study, the effect of water content on the mechanical behavior of interlayer soils under various coarse grain contents was investigated by monotonic triaxial tests. Three water contents of fine soil (wf = 17.6%, 10.6%, and 7.0%), five volumetric coarse grain contents (fv = 0%, 10%, 20%, 35%, and 45%) and three confining pressures (σ3 = 30, 60 and 120 kPa) were considered. Results showed that a decrease of wf led to an increase of shear strength and soil stiffness due to the effect of suction, and to an increase of dilatancy due to the aggregation of fine soils. Moreover, the variations of maximum deviator stress qmax, Young’s modulus E0, dilatancy angle ψ and friction angle φ with fv followed a bi-linear pattern for the three σ3 values, defining a characteristic volumetric coarse grain content fv-cha value for a given wf value: fv-cha ≈ 25%, 29% and 33% for wf = 17.6%, 10.6% and 7.0%, respectively. The fv-cha corresponded to the transition from a structure dominated by fine soils to a structure dominated by coarse grains. The increase of fv-cha with the decrease of wf could be attributed to the swelling and shrinkage of fines. While drying from optimum water content of fines wopt-f = 13.7% to a lower wf value, more coarse grains were needed to constitute the global skeleton due to the increase of the global volume of macro-pores resulted from the shrinkage of fine soils. By contrast, while wetting from wopt-f = 13.7% to a higher wf value, since the global volume of macro-pores decreased due to the swelling of fine soils, less coarse grains were required to constitute the global skeleton.