This study investigates the microstructure, phase composition evolution, and mechanical anisotropy of 321 stainless steel (SS) fabricated via laser powder bed fusion (LPBF). The influence of volume energy density on the morphology of molten pool and characteristics was investigated through experiments and simulations. Experimental results indicate that LPBF 321 SS predominantly consists of austenite with trace amounts of martensite. Through parameter optimization, nearly-fully-dense specimens with a relative density of similar to 99.9 % were prepared. It exhibits an excellent strength-plasticity balance surpassing that of castings/forgings. The ultimate tensile strength and elongation along the transverse direction reached 704 MPa and 61.5 %, respectively, while the longitudinal values were 625 MPa and 73.2 %. Grains oriented perpendicular to the building direction exhibit finer grains, higher proportion of low-angle grain boundaries and a lower Schmidt factor, all of which contribute to enhanced strength. Two substructures, cellular and columnar, were observed within the grains. During plastic deformation, the cellular substructure with dislocation walls effectively pinned dislocations and suppressed deformation twin propagation, while columnar substructures showed weaker blocking ability depending on their alignment. The higher cellular substructure content in the transverse direction contributed to greater tensile strength. These findings provide valuable insights for expanding the industrial production applications range of 321 stainless steel.