Synthetic biodegradable polymers including poly(lactic acid) (PLA) are attractive cell culture substrates because their surfaces can be micropatterned to support cell adhesion. The cell adhesion properties of a scaffold mainly depend on its surface chemical and structural features; however, it remains unclear how these characteristics affect the growth and differentiation of cultured cells or their gene expression. In this study, we fabricated two differently structured PLA nanosheets: flat and microgrooved. We assessed the growth and differentiation of mouse primary cultured cortical neurons on these two types of nanosheets after pre-coating with poly-D-lysine and vitronectin. Interestingly, prominent neurite bundles were formed along the grooves on the microgrooved nanosheets, whereas thin and randomly extended neurites were only observed on the flat nanosheets. Comparative RNA sequencing analyses revealed that the expression of genes related to postsynaptic density, dendritic shafts, and asymmetric synapses was significantly and consistently up-regulated in cells cultured on the microgrooved nanosheets when compared with those cultured on the flat nanosheets. These results indicate that microgrooved PLA nanosheets can provide a powerful means of establishing a culture system for the efficient and reproducible differentiation of neurons, which will facilitate future investigations of the molecular mechanisms underlying the pathogenesis of neurological disorders.