Patterning of electrospun nanofibers has recently attracted much attention for its usefulness in awide range of applications. This paper reports on generation of spatially defined nanofibrouspatterns by direct deposition of electrospun nanofibers onto a variety of insulating substrates. Itwas found that topographical features of different non-conducting substrates could be readilyreplicated by the electrospun nanofibers of interest. To elucidate the mechanism of nanofiberpatterning, we have systematically studied effects of the surface topography of non-conductingsubstrate (in particular the protrusion) and the electric forces associated with the electric field, onthe nanofiber deposition and assembly. By introducing the dielectric constant, it was identifiedthat the key factor that dictated the selective deposition and the formation of nanofibrous patternswill vary in different dielectric materials. If the dielectric constant of a particular non-conductivecollecting substrate is far greater than an identified critical range of 1.1797 ~ 1.3774, surfacetopography of the dielectric substrate will be the key factor that determines the deposition andthe arrangement of electrospun fibers. This contribution is believed to warrant further scientificunderstanding on the patterning mechanism of electrospun nanofibers, and to allow for design ofspecific and complex non-conductive substrate collectors for easy generation of patternednanofibrous architectures applicable in a variety of areas, such as tissue engineering,optoelectronic display, and so on.