Abstract: Water magnetite nanofluids for Polymer Electrolyte Membrane (PEM) fuel cell cooling application have been investigated. Nanofluid of water-magnetite (Fe3O4) has been synthesized using a two-step method. The particle size and its distribution, the stability and thermal conductivity of the nanofluid were characterized. The nanofluid is stable after 90 days (zeta potential value of 32.11 mV), and the measured thermal conductivity of the nanofluid at ambient temperature is 0.60 W/m.°C. The particles and nanofluid characterizations were used as the parameters in the analytical model to investigate the effect of particle diameter and volume fraction to the thermal conductivity of nanofluid and heat transfer in the PEM fuel cell. The analytical model suggested that the PEM fuel cell could produces an output power of 100 W and the heat that needs to be removed (cooling load) of 180 W, where 1×10−3 kg/s of nanofluid is required. The analytical model that used a particle diameter of 120 nm produces similar nanofluid’s thermal conductivity of 0.6 W/m.°C as the measurement. Less diameter particle improves the nanofluid’s thermal conductivity value. Higher volume fraction of 0.25 could enhances the nanofluid’s thermal conductivity value to 0.61 W/m.°C.