High mortalities and suppressed immune functions of farmed fish over winter are the universal problems in aquaculture. It is necessary to improve the immune response and disease resistance in the overwintering fish. A recent study suggested that repeated handling increased innate immune mechanisms and disease resistance in Senegalese sole. Therefore, the present study evaluated the hypothesis that appropriate repeated handling could compromise the immune depression and increase the disease resistance in gibel carp over winter. The experiment was executed in field net cages (2 m × 2 m × 2 m) from Dec. 4, 2012 to Apr. 2, 2013. Three cages with 50 fish per cage were randomly designed as the control group and did not receive any interfere over winter. The other three cages received repeated handling with an air exposure for 5 min every week during the experiment. Fish were not fed over winter. At the end of the trial, fish were challenged with Aeromonas hydrophila at a dose of 1.5 × 108 CFU ml−1. The results showed that no significant difference of final body weight was found between groups. The spleen and kidney somatic index increased in the control fish after bacterial challenge and showed a rising trend but not a statistical change in repeated handled fish. Plasma cortisol levels significantly increased in the control fish at 6 h post bacterial challenge and then declined. However, repeated handled fish did not show any significant change in plasma cortisol levels after challenge. The reduced inducement of heat shock protein 70 (HSP70) expressions by repeated handling was found in gibel carp post bacterial challenge. After overwintering, the repeated handled fish exhibited increased catalase (CAT) and superoxide dismutase (SOD) activities. Enhanced plasma CAT activities and reduced plasma malondialdehyde (MDA) contents were found in repeated handled fish over time against invading bacteria. Up-regulation of myeloid differentiation primary response gene 88 (MyD88) and interleukin 11 (IL11) was observed in repeated handled fish over time after bacterial challenge. The overexpression of IL11 was significantly reduced by repeated handling against invading bacteria compared to the control group. The present results implied that a MyD88-dependent signaling pathway was involved in the innate immune responses of gibel carp by repeated handling over winter against invading bacteria.