Long-term sperm storage in the female spermatheca allows Amphioctopus fangsiao to separate mating from fertilization, but the molecular responses of stored sperm remain unclear. We compared female-stored sperm (FS) with male sperm (MS) using Data-independent acquisition (DIA)-based quantitative proteomics and measured lactate content and mitochondrial membrane potential. A total of 2,169 differentially abundant proteins were identified, including 1,890 proteins with higher abundance and 279 with lower abundance in FS relative to MS. GO analysis identified 54 significantly enriched terms, including 37 related to sperm storage, while KEGG analysis identified 17 enriched pathways, including 8 associated with energy metabolism, stress responses, cellular survival, and reproductive signaling. PPI analysis of 868 well-annotated proteins identified 12 key proteins involved in energy metabolism, proteostasis, stress regulation, receptor-linked signaling, and calcium regulation. ENO1, PGM1, and PHGDH showed higher abundance in FS, whereas lactate content and mitochondrial membrane potential did not differ significantly between FS and MS. Lactate levels differed significantly between sperm and gill tissues. These findings indicate that long-term sperm storage involves coordinated proteomic remodeling that maintains metabolic flexibility, stress tolerance, and cellular stability while preserving sperm viability.