Anode-free sodium metal batteries (AFSMBs) with initial zero sodium anodes are promising energy-storage devices to achieve high energy density and low cost. The morphology and reversibility of sodium controls the cycling lifespan of the AFSMBs, which is directly affected by the separator. Here, we compared the sodium deposition and corresponding electrochemical behaviors under the influence of three commercial separators, which were celgard 2500, Al2O3-coated PP separator and glass fiber (denoting as 2500, C-PP and GF). Firstly, the reversibility of sodium plating/stripping was tested using half-cells, where coulombic efficiencies were stable at ~99.89% for C-PP and GF compare to 99.65% for 2500, indicating more dead sodium were formed for 2500. Then, the morphologies of deposited sodium were compared using optical microscopy. Compared to inhomogeneous sodium growth under 2500, C-PP obtained more flatter sodium layer with less height difference, attributing to the high mechanical strength of Al2O3 layer. Differently, we discovered that sodium was grown into pores in GF to form sodium particles with large active surface, which contacts with sufficient electrolytes and could be reversibly stripped. The reversibility of the sodium in GF were further verified using in-situ X-ray diffraction tests. Accordingly, cycling performance of AFSMBs were improved using C-PP and GF, where capacity retention after 120 cycles were 56.9%, 61.6% and 69.2% for 2500, C-PP and GF, respectively. Moreover, the AFSMB using 2 mAh cm-2 Na[Ni1/3Fe1/3Mn1/3]O2 as cathode with GF exhibiting excellent capacity of 117.61 mAh g-1 under high current density of 1C. Subsequently, in-situ EIS tests after/during charging/discharging process were further conducted to illustrate the enhancement of rate and cycling performance. This work demonstrates the effect of separators on the sodium deposition for higher irreversibility and stability, which could also offer insights for developing advanced separators to achieve high performance AFSMBs.