<p>Carbon-based materials are currently the primary commercial anode for lithium-ion batteries. Studies have shown that the specific surface area, pore size distribution, and heteroatom doping of carbon materials can affect their electrochemical performance. Previous investigations rarely explored the impact of carbon microcrystalline structures (such as interlayer spacing, microcrystallite size, and disorder degree) on the lithium storage capacity. Based on this issue, specifically designed carbon materials with an extremely small specific surface area and narrow pore size distribution were prepared from biomass. This unique structure effectively excludes the influence of pore structure on lithium storage properties, making it an ideal material for studying the effect of carbon microcrystalline structures on electrochemical properties. The combination of first-principles density functional theory (DFT) calculations, electrochemical experiments, and characterizations confirmed that the sample with the largest interlayer spacing, the highest <i>L</i>_<i>c</i> value, the smallest <i>L</i>_<i>a</i>, more vacancy defects, and a moderate amount of graphitic N demonstrated the highest reversible capacity, exceptional long-cycle stability, and superior rate capability. This work will provide theoretical guidance for the design of high-capacity carbon anodes with optimized microcrystalline structures for in lithium-ion batteries.</p> Graphical Abstract <p></p>