In order to investigate the effect of hold time on low cycle fatigue behavior of NT (normalized and tempered) 2 1/4Cr-1Mo steel at 723 K (0.4 Tm), strain controlled low cycle fatigue tests with three different tension-hold times, i.e. 900, 1800 and 3600 sec, were carried out. Fracture surface and microstructural change were examined to investigate the effect of hold time on the fracture process using optical, scanning electron and transmission electron microscopes (OM, SEM and TEM). Fatigue life was decreased and cyclic softening became larger as hold time increased. Judging from the examination by OM, SEM and TEM, hold time dependence of the cyclic softening and fatigue life is thought to be caused by the enhanced accumulated damage level during tension holding, not by creep effect.