The phenotype of reciprocal-crossing F1 was affected by cytoplasmic genome. Interestingly, no reproductive isolation was detected using Cucurbita maxima x Cucurbita moschata, but the sterility was observed using C. moschata x C. maxima (reciprocal cross). This phenomenon restricted the research on cytoplasm genetic effect of reciprocal-crossing in C. moschata and C. maxima as well as the transformation progress of cytoplasmic genome in C. moschata. In this study, MO (C. moschata, inbred line with disease resistance) and MA (C. maxima, core parent line) were selected to make inter-specific crosses. The immature embryo of MO×MA was cultured and the suitable cultural period, sterilizing procedure and culture medium were selected. The hybrid reliability of 6 regenerated plants (E0) was identified by co-dominant SSR markers. The results showed that the suitable cultural period of the immature embryo of MO×MA was 25 days post pollination. Under the lab condition, the immature embryos were removed from seeds carefully and soaked using 2% NaClO for 5 min to remove the endotesta. Then the embryos were moved into super-clean worktable, surface sterilized in 75% (v/v) ethanol for 30s, followed by immersion in 2% NaClO for 5 min, and rinsed with sterile distilled water 3 times. This sterilization pipeline showed the lowest contamination rate (39%), highest germination rate (24%) and convenient operation. The immature embryos on the No.8 culture medium (MS + 3.0 mg/L 6-BA + 0.5 mg/L 2,4-D + 0.2 mg/L NAA + 20g/L sugar + 0.68% agar) resulted in the highest embryos germination rate of 25%. The effect of 3-day initial incubation in dark on the MO×MA immature embryo culture was also tested, but no visible difference was detected. By marker-assisted identification using 23 and 8 co-dominant SSR markers, these six E0 regenerations of MO×MA were the real hybrid and could be used for further cytoplasm genome transfer of C. moschata.