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 interest is the investigation of the morphology and function of the central nervous system 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 was downward mounted on a rotating stage at an angle of 45°. After the exposure was repeated three or more times, maintaining the same exposure plan but rotated by the same angle, a photoresist was developed to produce a 3D porous template. Afterwards, 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. A culture test was conducted using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenous cell infiltration and 3D attachment. As expected, a much higher cell number was found in the 3D PDMS lattices compared to the 2D culture. We also found a higher neuron-to-astrocyte ratio and a higher degree of cell ramification in the 3D culture compared to the 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.