Pumpkin is widely used as a functional food for people and rootstock for other Cucurbitaceae crops. Se-enriched pumpkin can be used as an important Se source for humans. However, the boundary between beneficial and phytotoxic levels of Se is narrow. Here, effects of selenite with different concentrations (2–80 μM) on pumpkin growth and Se accumulation in pumpkin seedlings were explored. Our results revealed that Se accumulations in both shoots and roots of C. moschata were closely related to selenite doses. Selenite seemed to have no positive effects on C. moschata growth. The root activity increased at 2–20 μM of selenite, but significantly decreased at higher doses. High concentrations of selenite (above 40 μM) resulted in serious O2•– accumulation, growth inhibiting and even symptoms of toxicity, which could be alleviated by melatonin. Transcriptome analysis revealed that a series of defense mechanisms, including Ca2+ signaling, ET and JA signaling, antioxidant defense systems, MAPK cascade, proteostasis protection, could be activated in roots of C. moschata in response to 40 μM selenite. Additionally, we identified some important genes probably involved in Se absorption and conversion in C. moschata, such as PHT, SULTR, APS, APK, APR, SiR, GR, and CS genes. These results may help our understanding of the physiological and molecular mechanisms of Se accumulation and tolerance to Se toxicity in C. moschata, which may provide genetic resources and a theoretical basis for the breeding and cultivation of Se-rich pumpkin or rootstocks.