Abstract: Overwintering is a special stage during metabolism and immunity of the fish are suppressed, which often induces high mortality rate in aquaculture. Therefore, it is meaningful to elucidate the immune inhibition in overwintering farmed fish and try to reduce the mortality rate. Microalgae containing highly unsaturated fatty acids and other nutrients, and the previous studies showed that dietary microalgae could enhance the immunity of fish. It was reported that replacement of fishmeal by Arthrospira platensis improved the immunity of gibel carp partly via the toll like receptor 2 (TLR2) pathway. The Toll-like receptor (TLR) signaling pathway including the myeloid differentiation factor 88 (MyD88)-independent and MyD88-dependent pathways is a key pathway involved in the innate immune response. Gibel carp (Carassius gibelio) is a common economic species in China, and it must face the high mortality rate during winter. Aeromonas hydrophila is a common pathogenic bacterium which could induce high mortality in gibel carp over winter. Scenedesmus ovalternus HSJ296 was reported to have relatively high α-linolenic acid, but there were few reports about its application in aquaculture feed. To investigate the effects of dietary S. ovalternus on the metabolism and disease resistance of overwintering gibel carp (C. gibelio var. CAS Ⅴ), during the early overwintering period, gibel carp initial body weight of (109.24±0.23) g were fed with two diets: DS0 (containing 0 S. ovalternus) or DS4 (containing 4% S. ovalternus) for 4 weeks. During the late overwintering period, all fish were left unfed and then challenged with A. hydrophila. The growth performances, immunity, and disease resistance against A. hydrophila infection in overwintering gibel carp were measured. Plasma immunological and antioxidant indicators including the activities of myeloperoxidase (MPO), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD), and the contents of interleukin 2 (IL2), tumor necrosis factor α (TNFα), complement 3 (C3), immunoglobulin M (IgM), and malondialdehyde (MDA) were tested. The mRNA levels of immune-related genes involved in TLR signaling pathways in the head kidney of gibel carp were tested. The results showed that dietary S. ovalternus had no effects on the growth or survival rate of gibel carp after the challenge (P>0.05). The activities of MPO were similar between the two groups (P>0.05) but increased in DS4 group after challenge (P<0.05). The mRNA levels of MyD88 were higher in the DS4 group than that fed the DS0 diet after bacterial challenge (P<0.05). The bacterial challenge induced toll like receptor 4 (TLR4), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP) and TIR-domain-containing adapter-inducing interferon β (TRIF) in fish fed DS4 diet (P<0.05), but not in fish fed DS0 diet (P>0.05). Overall, S. ovalternus might activate both MyD88-dependent and -independent TLR signaling pathway in gibel carp to against the bacterial challenge. The present study could provide the data of improving immunity and disease resistance in overwintering gibel carp by dietary S. ovalternus, which offers new insights into improving the overwinter survival of fish.