Magnetic particle imaging (MPI), using magnetite nanoparticles (MNPs) as tracer material, shows great promise as a platform for fast tomographic imaging. To date, the magnetic properties of MNPs used in imaging have not been optimized. As nanoparticle magnetism shows strong size dependence, the authors explore how varying MNP size impacts imaging performance in order to determine optimal MNP characteristics for MPI at any driving field frequency f0. Monodisperse MNPs of varying size were synthesized and their magnetic properties characterized. Their MPI response was measured experimentally using a custom-built MPI transceiver designed to detect the third harmonic of MNP magnetization. The driving field amplitude H0 = 6 mT micro0(-1) and frequency f0 = 250 kHz were chosen to be suitable for imaging small animals. Experimental results were interpreted using a model of dynamic MNP magnetization that is based on the Langevin theory of superparamagnetism and accounts for sample size distribution and size-dependent magnetic relaxation. The experimental results show a clear variation in the MPI signal intensity as a function of MNP diameter that is in agreement with simulated results. A maximum in the plot of MPI signal vs MNP size indicates there is a particular size that is optimal for the chosen f0. The authors observed that MNPs 15 nm in diameter generate maximum signal amplitude in MPI experiments at 250 kHz. The authors expect the physical basis for this result, the change in magnetic relaxation with MNP size, will impact MPI under other experimental conditions.