The conventional railway substructure in France was built by emplacing ballast directly on sub-grade. Over years of operation, the inter-penetration of ballast and sub-grade 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 interlayer soil, assessing the influence of fine particles content on the hydraulic behavior of interlayer soil is of importance. To this end, the hydraulic behavior of an interlayer soil taken from Sénissiat (near Lyon, France) was investigated using two infiltration columns, a large-scale column equipped with tensiometers and 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 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 suction effect. By contrast, in saturated state, the value for the interlayer soil with 10% fine particles added was found higher, suggesting that in this case the hydraulic conductivity is mainly governed by the water transfer through macro-pores.