Mesocarp color is an important agronomic trait of Cucurbita maxima and is determined mainly by the contents and compositions of the carotenoids. The two inbred lines with significant differences in fruit flesh color were used in the study, the orange\'312-1\' and white \'98-2\'. Changes in seven carotenoid contents and compositions in the flesh of fruit produced by inbred lines\'312-1\' and\'98-2\' were analyzed during fruit development. The expression of eight key carotenoid biosynthesis genes in the fruit flesh were investigated during five fruit development stage in two inbred lines. As the flesh color intensified, the orange flesh color of\'312-1\' was determined mainly by the increased contents of lutein,β-carotene and zeaxanthin and the lack of carotenoid accumulation led to the formation of white flesh in\'98-2\' fruit. The expression of the LCY-e and CHYb genes was significantly stronger in\'312-1\' than in\'98-2\', and their expression was strongly correlated with lutein andβ-carotene contents during fruit development. This study provides a deep understanding of the molecular mechanism of carotenoid biosynthesis in fruit flesh and provides a basis for additional studies on the highly refined improvement of squash quality.