Abstract: This study aimed to systematically determine the composition of freeze-dried blackberry powder (FDBP) and investigate its protective effects against germ cell damage induced by oxidative stress. FDBP was prepared using processes with or without blackberry seed removal. Quantitative analysis was conducted on the contents of key bioactive components including amino acids, polyphenols (anthocyanins), vitamins (E, folate), and trace elements in FDBP. An oxidative stress model was established using the mouse spermatocyte cell line GC-2spd treated with H₂O₂ to explore the protective effect of FDBP against oxidative stress-induced germ cell damage. The results indicated that both types of FDBP were rich in amino acids such as alanine, glutamic acid, aspartic acid, and arginine, and contained trace elements including magnesium, iron, zinc, and selenium. Among them, the thawed and deseeded FDBP (FDBP-1) had higher contents of anthocyanins, vitamin E, and folate compared to the freeze-dried and superfinely ground FDBP (FDBP-2), measuring 6.89±0.13 mg/g, at 85.54±4.79 μg/g, and 22.94±2.68 μg/g, respectively. FDBP-1 demonstrated stronger antioxidant activity than FDBP-2, as evidenced by its lower IC₅₀ values for scavenging DPPH radicals (126.60±44.78 μg/mL) and ABTS⁺ radicals (29.84±10.84 μg/mL). Under oxidative stress conditions, pretreatment with FDBP-1 significantly increased the viability of GC-2spd cells by 28%. Furthermore, it effectively upregulated the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while reducing malondialdehyde (MDA) levels. In conclusion, FDBP is rich in various bioactive compounds and possesses excellent in vitro antioxidant capacity. It effectively protects germ cells against oxidative damage by enhancing the cellular endogenous antioxidant defense system. This study provides a crucial scientific basis for developing FDBP-based functional foods or ingredients with reproductive health-protective effects, demonstrating significant application potential in the food industry and nutritional health sectors.