In the present study, Cu2O films were deposited on a glass substrate via RF (radio frequency) magnetron sputtering under substrate temperature conditions that ranged from room temperature (RT, 25 °C) to 400 °C. The structural, compositional, and optical properties of the Cu2O films were analyzed in relation to the experimental variables by applying various measurement methods. The substrate temperature was a crucial factor in shaping the structural, compositional, and optical properties of the Cu2O films that were synthesized via RF-magnetron sputtering. Our findings revealed that the Cu2O films exhibited a cubic structure, which was confirmed by XRD analysis. Specifically, the (111) and (200) planes showed different trends with respect to the substrate temperature. The intensity of the (111) peak increased at 250 °C, and above 300 °C, the preferred orientation of the (111) plane was maintained. The grain size, which was determined via FE-SEM, displayed a positive correlation with the substrate temperature. Additionally, XPS analysis revealed that the binding energy (BE) of the Cu2O film sputtered at 400 °C was similar to that which was previously reported. Notably, the as-grown Cu2O film demonstrated the highest transmittance (15.9%) in the visible region, which decreased with increasing substrate temperature. Furthermore, the energy band gap (Eg) of the Cu2O films remained constant (2.51 eV) at low substrate temperatures (25 °C to 200 °C) but exhibited a slight increase at higher temperatures, reaching 2.57 eV at 400 °C.