The growing interest in Na-ion batteries as a “beyond lithium” technologies for energy storage drives the research for high-performance and environment-friendly materials. Na2Ti3O7 (NTO) as an eco-friendly, low-cost anode material shows a very low working potential of 0.3 V vs. Na+/Na but suffers from poor cycling stability, which properties can be significantly influenced by materials synthesis and treatment. Thus, in this work, the influence of the calcination time on the electrochemical performance and the reaction mechanism during cycling were investigated. NTO heat-treated for 48 h at 800 °C (NTO-48h) demonstrated enhanced cycling performance in comparison to NTO heat-treated for only 8 h (NTO-8h). The pristine material was thoroughly characterized by X-ray diffraction, laser granulometry, X-ray photoelectron spectroscopy, and specific surface area measurements. The reaction mechanisms induced by sodiation/desodiation and cycling were investigated by operando XRD. Electrochemical impedance spectroscopy was used to evidence the evolution of the solid electrolyte interface layer (SEI) and modification of charge transfer resistances as well as the influence of cycling on capacity decay. The evolution of the crystallographic structure of NTO-48h revealed a more ordered structure and lower surface contamination compared to NTO-8h. Moreover, the residual Na4Ti3O7 phase detected after the sodium extraction step in NTO-8h seems correlated to the lower electrochemical performance of NTO-8h compared to NTO-48h.