Temperature is a fundamental physical quantity that governs every biological reaction within living cells, and intracellular temperature reflects cellular thermodynamics and function. In medical studies, the cellular pathogenesis of diseases (e.g., cancer) is characterized by extraordinary heat production. Therefore, intracellular temperature imaging should promote better understanding of cellular events and the establishment of novel diagnoses and therapies. We have demonstrated the first intracellular temperature imaging based on a fluorescent polymeric thermometer and quantitative fluorescence imaging techniques. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.2 °C, respectively. We first performed tracking of the averaged temperature of a single whole cell and showed that the averaged temperature of single COS7 cells increased by 0.98 °C upon mitochondrial uncoupling. Next, we developed a novel method to visualize intracellular temperature distribution, which indicated that the nucleus of a COS7 cell showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. Furthermore, imaging of intracellular thermogenesis was also investigated: we could observe the local temperature change provoked by both endogenous heat production from mitochondria and external heating using infrared laser. These results demonstrate an intrinsic connection between intracellular temperature and cell function. Thus, our intracellular temperature imaging will provide insights into the regulatory mechanisms of intracellular signaling.