To reveal the intrinsic strengthening mechanism in Ni-based single crystal superalloy, the interaction between matrix dislocations and interfacial misfit dislocation networks was modeled in this contribution via molecular dynamics (MD) method. Our results show that the role of interfacial dislocation networks is very complex. On the one hand, the interfacial dislocation networks can act as dislocation sinks to absorb/accommodate the matrix dislocations. During the accommodation process of matrix dislocation by the networks, both the interfacial Lomer–Cottrell locks and a[100] dislocation junctions are formed, which stabilize and strengthen the interfacial dislocation networks. On the other hand, the interfacial dislocation networks can provide dislocation pins to prevent the matrix dislocations from cutting into the γ′ precipitate. These matrix dislocation segments pinned at the phase interface can serve as Frank–Read sources with their length being about half of the dislocation network spacing, providing an explanation for the effect of dislocation network spacing on the creep strength of the Ni-based single crystal superalloy.