The oxygen content of precursors plays a key role in regulating the structural stability and microstructures of hard carbon anodes towards sodium-ion batteries, but this is often neglected in the previous reports. Herein, we select the esterified starch as a model precursor and quantitatively regulate its oxygen content by low-temperature hydrogen reduction. Through the correlation analysis of oxygen content changes and the microstructural information of derived hard carbons, we find that decreasing the oxygen content of precursors but guaranteeing the stability of crosslinking structures can promote the closure of open pores and the orientated alignment of carbon layers at relatively low carbonization temperature (1100 °C). The optimal sample exhibits a low specific surface area of 2.96 m2 g−1 and high proportion of pseudo-graphitic domains. The structural advantages of the hard carbon contribute to a high reversible sodium storage capacity of 369.8 mAh g−1 with an initial Coulombic efficiency (ICE) of 82.5% at 20 mA g−1. Furthermore, in-situ Raman spectroscopy results demonstrate that pseudo-graphitic structures, with large interlayer spacing, provide sufficient diffusion channels for Na+ ions intercalation and pore-filling. This work provides new insights for the microstructure regulation and design of other precursor-derived hard carbons.