21-6-9 stainless steel manufactured by powder bed fusion (PBF-LB/M) is expected to be used for aerospace parts due to its high strength and elevated temperature durability, coupled with the possibility to manufacture complex shapes. Defects, such as pores and lack of fusion (LoF), can be formed in PBF-LB/M manufactured material, which can be detrimental to important dynamic mechanical properties like fatigue strength. However, hot isostatic pressing (HIP) has shown great potential in closing and healing such defects. In this study, an investigation of the effects of HIP treatment on the low-cycle fatigue (LCF) properties and microstructure of PBF-LB/M manufactured 21-6-9 material was performed. It was found that the effects of HIP treatment are similar to that of solution heat treatment regarding grain coarsening and grain sensitization. In addition, HIP successfully closed the pores, reducing the defect density. The stress relief heat-treated (SR) specimens exhibited lower LCF strength than wrought material, but after HIP treatment, LCF strength was equal to wrought material. The HIP treatment consequently improved the strain-controlled LCF strength but decreased the tensile strength and hardness around 30%.