Titanium-stabilized AISI 321 material (UNS S32100) is generally preferred in the pressure vessel industry as it is not sensitive to intergranular corrosion. In critical applications, the fatigue behavior of weld seams is among the most stringent requirements. The microstructural characteristics and fatigue performance double-side-welded AISI 321 plate having 6 mm thickness was evaluated in this work. AISI 321 was welded with double-side-gas tungsten arc welding (DS-GTAW) process. The fatigue behavior was examined with a loading ratio of R = 0.1 and frequency of 15 Hz for two different specimens: base metal (BM) and weld metal (WM). Monotonic tensile results shows the improved tensile properties of WM compared to BM samples. The fatigue strength of WM (150 MPa) was 25% higher than that of BM (120 MPa) specimen and is attributed to the increase in ferrite volume along with dendritic microstructure. The change in the fraction of low angle grain boundaries (LABs) and high angle grain boundaries (HABs) improved the tensile and fatigue properties. The stress amplitudes influenced the degree of striations in the BM and WM. Final fracture surfaces were characterized with dimples and microvoids, revealing the ductile mode of fatigue fracture. The fatigue rupture surfaces of BM and WM samples at different stress regimes are discussed.