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In this study, three kinds of round-shaped pitch-based graphite fiber with different microstructural features (crystallinity and carbon layer orientation) were fabricated by melt-spinning, preoxidation, carbonization and graphitization. The morphology, crystalline size and carbon layer orientation of carbon fibers from different pitch precursors and spinning rates were characterized through X-ray diffraction, scanning electron microscopy and transmission electron analyses. The correlation of the electrochemical performance and microstructure of graphite fibers as anode materials for lithium-ion batteries was investigated. The results suggest that large-diameter anisotropic graphite fibers (L-AF3000) with a radial texture of the transverse section are more favorable for lithium intercalation storage. The discharge capacity of L-AF3000 is 319.1 mAh∙g⁻¹ at 0.1 <i>C</i> (current density). Nevertheless, the capacity drops to 209.9 mAh∙g⁻¹ at a high current density of 1 <i>C</i>, and the capacity retention is only 82.2% over 100 cycles at 0.1 <i>C</i>. Small-diameter anisotropic graphite fibers (S-AF3000) with a spiral-shaped wrinkle texture of the transverse section possess discharge capacities of 284.1 mAh∙g⁻¹ at 0.1 <i>C</i> and 260.2 mAh∙g⁻¹ at a high current density of 1 <i>C.</i> Meanwhile, the best capacity retention of the fibers is 101.6% over 100 cycles at 0.1 <i>C</i>. The results suggest that the disordered carbon layers in S-AF3000 can retain the structural integrity of fibers as anode material for lithium-ion batteries and thus obtain excellent cycle stability. In addition, larger crystalline sizes of fibers correspond to higher discharge capacity, and a smaller diameter is beneficial to the fast insertion and extraction of lithium-ion in fibers.