Neonatal and stem cell-derived cardiomyocytes differ from adult heart cells in their structure and function, limiting their use in pharmacological studies. Establishment of anisotropic cultures can improve the functional properties of cardiomyocytes; however, the mechanism that links functional improvement with ultrastructural reorganisation is unclear. Microtubules act as a link between mechanical stimuli and functional response in cardiac cells but whether they are affected during cellular alignment in culture is unknown. In this study neonatal rat ventricular myocytes were cultured on micropatterned Parylene C constructs, with alternating hydrophilic-hydrophobic parallel lines, to induce cellular alignment. The cells formed an anisotropic monolayer, those on the 10μm thick constructs were more constrained within the lines and cylindrical in shape compared with the cells on 2μm thick constructs (p<0.01). The cells were field stimulated at 2Hz to produce calcium transients which were optically mapped using Fluo-4. Compared with unaligned cultures, cells on the aligned constructs demonstrated a significant decrease in time to peak (tp unaligned=129±7ms, tp aligned=82±4ms, p<0.001, n=34,40 respectively) and 90% decay (t90 unaligned=453±17ms, t90 aligned=270±6ms, p<0.001, n=34,40 respectively). Within the aligned constructs, Parylene's thickness was found to affect calcium cycling parameters. Cells cultured on 2μm thick constructs showed a significant decrease in tp compared to 10μm thick constructs (tp 2μm thick=123±2ms, tp 10μm=107±2ms, p<0.001, n=326,197 respectively) and t90 (t90 2μm=275±1ms, t90 10μm=261±2ms, p<0.001, n=326,197 respectively). Microtubules were identified by immunofluorescence for alpha-tubulin, indicating a significant increase in their density on the aligned constructs compared to unaligned for both thicknesses (2μm thick:area covered aligned=63.1±1.4%, area covered unaligned=55.7±1.3%, p<0.01, n=180 for both; 10μm thick:area covered aligned=68.0±1.5%, area covered unaligned=54.4±1.3%, p<0.001, n=180,150 respectively). Also, cells on the 10μm thick micropatterned constructs had an increased microtubule density compared to the 2μm thick constructs (area covered 10μm thick=68.0±1.5%, area covered 2μm thick 63.1±1.4%, p<0.01, n=180). These results indicate that reorganisation of cardiomyocyte morphology with increased anisotropism causes enhanced calcium cycling, which is associated with increased microtubule density. Further study is required to determine the molecular mechanisms involved and whether microtubules can be targeted directly to favour cardiac cell maturation in culture.