The Spitzer Space Telescope was used to study the mid-to far-infrared properties of NGC 300 and to compare dust emission to Hα to elucidate the heating of the interstellar medium (ISM) and the star formation cycle at scales smaller than 100 pc. The new data allow us to discern clear differences in the spatial distribution of 8 μm dust emission with respect to 24 μm dust and to H II regions traced by Hα light. The 8 μm emission highlights the rims of H II regions, and the 24 μm emission is more strongly peaked in star-forming regions than 8 μm. We confirm the existence and approximate amplitude of interstellar dust emission at 4.5 μm, detected statistically in Infrared Space Observatory (ISO) data, and conclude it arises in star-forming regions. When averaging over regions larger than∼ 1 kpc, the ratio of Hα to aromatic feature emission in NGC 300 is consistent with the values observed in disks of spiral galaxies. The mid-to far-infrared spectral energy distribution of dust emission is generally consistent with pre-Spitzer models.