Z3CN20.09M cast duplex stainless steel (CDSS) has been used for primary coolant water pipes in pressurized water reactors due to its excellent mechanical properties. Such pipes operate at an elevated service temperature (~320 °C) and experience issues of thermal aging embrittlement. In situ tensile tests were conducted to investigate the deformation mechanisms of Z3CN20.09M CDSS after long-term thermal aging at 475 °C for up to 2000 h in both optical microscope and scanning electron microscope at 320 °C. For the 320 °C tests, the tensile stress and other mechanical properties, e.g. the yield stress and the ultimate tensile strength, increase during the thermal aging process and recover to almost the same level as the unaged condition after annealing heat-treatment, which is caused by the formation and dissolution of precipitation during aging and anneal heat-treatment, respectively. For the slip mechanism, straight slip lines form first in the austenite phase. When these slip lines reach the austenite/ferrite interface, three kinds of slip systems are found in the ferrite phase. During the fracture process, the austenite phase is torn apart and the ferrite phase shows a significant elongation. The role of the ferrite phase is to hold the austenite matrix, thus increasing the tensile strength of this steel.