Graphene oxide(GO) has high thermal conductivity and strong hydrophilicity, which can significantly improve the heat transfer performance of fluids. In this study, effects of GO nanofluids on the start-up and heat transfer were investigated in a pulsating heat pipe(PHP) through experiment. The PHP consists of a thin copper tube, which was bent into a closed loop with 3 turns in the evaporator and condenser respectively. It was operated in a vertical bottom heating mode, i.e., the evaporation section was in the bottom with an electrical heating power in the range of 10—105 W, while the condenser section was in the top with the air forced cooling. The nanofluid was prepared by dissolving GO nanoparticles into the deionized water, with different mass fraction range of 0.02%—0.11%, and the filling ratio was kept constant about 50%. Results showed that adding appropriate GO nanoparticles could effectively improve the start-up performance, when compared with the pure water PHP. For the concentrations of 0.05% and 0.08%, the PHP could start up more easily and smoothly, in specifically, the start-up temperature reduced about 28.6℃ (33.9%) and 26.2℃ (31.1%) respectively, meanwhile the start-up time shortened about 320 s (19.5%) and 304 s (18.5%) respectively. As for the heat transfer enhancement effect, it was related to the concentration of GO nanoparticles and heating power of the PHP. There existed an appropriate concentration and heating power range (i.e., w=0.02%—0.08%, Q=20—105 W), which could reduce the thermal resistance of GO/water PHP by 18.6%—57.1% when compared with that of pure water. For the concentration range of 0.02%—0.08%, the improvement of thermal performance first increased and then gradually decreased with the increase of heating power. At the case of 30 W, the thermal performance could be improved by 46.1%—57.1%. When the concentration was relatively higher (e.g., w=0.11%), addition of GO nanoparticles could not improve the start-up and heat transfer performance of PHP, and might even worsen the performance. Finally, an empirical correlation was obtained to predict the thermal performance of the GO/water PHP based on the dimensionless combination of Ku, Bo, Mo, Pr, Ja*.