Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interesting is to investigate the morphology and function of the central nervous system (CNS) cultured using such scaffolds. Here, we used an elastomer, polydimethylsiloxane (PDMS), to produce lattice-type scaffolds from a photolithography defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist and downward mounted on a rotating stage at angle of 45°. After exposure for three or more times keeping the same exposure plan but rotated by the same angle, the photoresist was developed to produce a 3D porous template. Afterward, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. Culture test has been done using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenously cell infiltration and 3D attachment. As expected, a much higher cell number was found in 3D PDMS lattices than in 2D culture. We also found a higher neuron to astrocyte ratio and a higher degree of cell ramification in 3D culture compared to 2D culture, due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies.