In winter, fish have suppressed immune functions and are susceptible to bacteria or virus which may lead to a high mortality. It is necessary to improve the immune response and disease resistance for overwintering fish. A recent study suggested that repeated handling increased the innate immune mechanisms and disease resistance in Senegalese sole. Therefore, the present study tested the hypothesis that appropriate repeated handling could compromise the immune depression and increase the disease resistance in channel catfish over winter. The experiment was carried out in field cages from Nov. 2012 to April 2013. Before the experiment, 35 fish with an average weight of 188.4 g were randomly assigned to one of six cages (2 m × 2 m × 2 m). Three cages were designed as the control group and did not receive any interfere. Fish in the other three cages received a weekly repeated handling of an air exposure for 5 min. Fish were not fed over winter. At the end of the trial, fish were challenged with Aeromonas hydrophila at a dose of 5.6 × 10(8) CFU ml(-1). The results showed that final body weight was not changed between groups. The spleen somatic index increased while the head kidney somatic index decreased in the unhandled fish after bacterial challenge, but these indices were not significantly changed in the repeated handled fish. Plasma cortisol levels in the control fish were induced at 6 h post challenge and then declined to the normal levels. However, plasma cortisol levels in the repeated handled fish did not show any significant change after bacteria challenge. The reduced inducement of heat shock protein 70 (HSP70) expression by repeated handling was observed in fish post bacterial challenge. After overwintering, repeated handled fish exhibited increased catalase (CAT) activities and reduced malondialdehyde (MDA) contents. Plasma total antioxidant capacity (TAOC), CAT and superoxide dismutase (SOD) activities of channel catfish were enhanced by repeated handling post bacterial challenge. The enhanced up-regulation of Interleukin 8 (IL8), IL1β-a, IL1β-b together with the immune related genes of Toll-like receptor 2 (TLR2), TLR3, nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 by repeated handling was found in catfish after bacterial challenge. The present results indicated that a combination of signaling pathways through TLRs and NODs was involved in the innate immune response of the overwintering repeated handled channel catfish against invading bacteria.