AbstractEngineered culture substrates with chemical neurite guidance cues have been used for studying the mechanism of axon pathfinding at cellular level. In this study, we designed a novel poly‐l‐lysine (PLL) micropattern (“pinstripe micropattern”) to investigate how the same biomolecules with slightly different surface concentration can affect in vitro neuronal growth. The pinstripe micropattern was fabricated by stamping PLL on a PLL‐coated glass coverslip, which resulted in denser PLL lines and a less‐dense PLL background. There were two effects of the substrate on cultured primary hippocampal neuron: neurite initiation and growth cone turning. Although the whole surface was permissive for neurite outgrowth, we observed that the growth direction of neurites had a strong tendency to follow the stamped PLL line patterns with PLL background. However, the micropattern did not affect the spreading of cell body on the substrate. According to these investigations, we concluded that the PLL pinstripe pattern with PLL background, which had the step difference of polylysine concentrations, would be very useful for designing novel cell assays for the investigation of neurite guidance mechanisms, and suggested it as a new design method for controlling the direction of neurite growth on in vitro neural network. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2731–2739, 2015.