This study investigated the effects of different overwintering temperatures (8, 12, 16, and 20 °C) on basic nutritional composition and non-volatile flavor substances, including free amino acids and free nucleotides of edible tissues (gonad, hepatopancreas, and muscle) of female mud crab Scylla paramamosain after eight-week of overwintering. The results showed that the overwintering process and temperature did not affect the basic nutritional composition of the gonad (P > 0.05). However, with the increase in temperature, the lipid content of hepatopancreas decreased and was accompanied by a rise in moisture content (P < 0.05). In muscle, moisture content increased significantly, and crude protein content decreased significantly after overwintering at 20 °C (P < 0.05). During the overwintering process and with the increase in temperature, free amino acids and free nucleotides decreased considerably (P < 0.05). In addition, each tissue had a high content of total sweet amino acids (SAA) under low overwintering temperatures. However, the contents of total umami amino acids (UAA) and equivalent umami concentration (EUC) increased significantly, accompanied by the decrease of total bitter amino acids (BAA) with the rising temperature (P < 0.05). To sum up, overwintering under high temperature was beneficial to improve the umami characteristic and reduce the bitter characteristic, but it would cause the crabs to lose and consume proteins and lipids. However, overwintering at low temperature had higher nutrients and sweet characteristic. Therefore, overwintering at 8–12 °C is more conducive to improving the nutritional quality and flavor of S. paramamosain.