The objective of this study is to investigate the typical failure mode and to obtain the stress range versus number of cycles to failure (S-N) data of MARK-III type liquefied natural gas (LNG) insulation system under the fatigue loading at actual cryogenic environment. A systematic experimental research is carried out for the assessment of the fatigue strength of MARK-III insulation system at cryogenic temperature. Three different types of test specimens are tested for the evaluation of fatigue performance of MARK-III insulation system. Test specimens are determined considering the fatigue vulnerable locations such as mastic area, slit area, and top bridge pad area inside the actual LNG cargo tanks. All test specimens are fabricated as close as possible to the actual yard practice. A series of fatigue test results is represented as S-N curves. Cyclic fatigue loadings were carefully considered similar to the actual sloshing loads. The effect of sloshing impacts is considered by selecting the stress ratio . The load levels have been determined based on the ultimate strength of reinforced polyurethane foam as 12.2 bars. Different cryogenic temperatures are employed according to the test locations in consideration of temperature gradient within the insulation system. All test results including fatigue life, as well as failure locations of MARK-III insulation system at cryogenic temperatures, are reported and compared with those at room temperature. Consistent S-N curves of MARK-III insulation system at both room and cryogenic temperatures are obtained and compared. The slopes of S-N curves from both fatigue test results are observed to be almost identical, and the fatigue strengths are found to exhibit similar trend. The results from this research can be used for the fatigue assessment of the LNGC insulation system, as well as a design guideline of LNG CCS at cryogenic temperature.