The conventional railway substructure in France was built by emplacing ballast directly on subgrade. Over years of operation, the interpenetration of ballast and subgrade created a soil layer between them. Under different conditions, this naturally formed layer, namely interlayer, can contain different quantities of fine particles, becoming more or less sensitive to changes in water content. As the water content changes are governed by the hydraulic behavior of the interlayer soil, assessing the influence of fine particle content on the hydraulic behavior of interlayer soil is of importance. To this end, the hydraulic behavior of an interlayer soil taken from Senissiat (near Lyon, France) was investigated using two infiltration columns, a large-scale column equipped with tensiometers and a time domain reflectometer (TDR) for suction and volumetric water content measurements, respectively, and a smaller column equipped with high-capacity tensiometers only. Different fines contents were considered and wetting-drying cycles were applied to the soil specimens. The hydraulic conductivity was determined by applying the instantaneous profile method. The results obtained showed that (i) hysteresis exists for both the soil water retention curve and the hydraulic conductivity changes with suction; (ii) the effect of wetting-drying cycles is insignificant; (iii) adding 10% fine particles to the natural interlayer soil gives rise to changes in the soil water retention curve but does not induce significant changes in hydraulic conductivity; (iv) the unsaturated hydraulic conductivity of interlayer soil with 10% fine particles added is close to that of soil sieved at 2 mm, suggesting that the unsaturated hydraulic conductivity of interlayer soil is mainly governed by fine particles through the suction effect. By contrast, in a saturated state, the value for the interlayer soil with 10% fine particles added was found to be higher, suggesting that in this case the hydraulic conductivity is mainly governed by the water transfer through macropores.