Nucleic acids can be unfolded either by temperature, such as in UV melting, or by mechanical force using optical tweezers. In UV melting experiments, the folding free energy of nucleic acids at mesophilic temperatures are extrapolated from unfolding occurring at elevated temperatures. Additionally, single molecule unfolding experiments are typically performed only at room temperature, preventing calculation of changes in enthalpy and entropy. Here we present temperature controlled optical tweezers suitable for studying folding of single RNA molecules at physiological temperatures. Constant temperatures between 22°C and 42°C are maintained with an accuracy of 0.1°C, whereas the optical tweezers display a spatial resolution of ∼1 nm over the temperature range. using this instrument, we measured the folding thermodynamics and kinetics of a 20-base-pair RNA hairpin by force-ramp and constant force experiments. Between 22oC and 42oC, the hairpin unfolds and refolds in a single step. Increasing temperature decreases the stability of the hairpin and thus decreases the force required to unfold it. The equilibrium force, at which unfolding and refolding rates are equal, drops ∼1 pN as temperature increases every 5oC. At each temperature, the folding energy can be quantified by reversible work done to unfold the RNA and from the equilibrium constant at constant forces. Over the experimental temperature range, the folding free energy of the hairpin depends linearly on temperature, indicating that ΔH is constant. The measured folding thermodynamics are further compared with the nearest neighbor calculations using Turner's parameters of nucleic acid folding energetics. Values of ΔS are comparable, however, ΔH from the two approaches are significantly different. The origin of such a difference will be further discussed.