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Autophagy is a highly conserved process in eukaryotic cells that sequesters intracellular components in the autophagosome for degradation by lysosomal enzymes. Western blot (WB) and immunofluorescence assay for LC3II were applied to detect autophagy modulation in the RTgill-W1 cell line. Two percent fetal bovine serum (FBS), 1M deoxynivalenol (DON) and 50M chloroquine (CQ) significantly (p < 0.05) activated, inhibited and blocked autophagy, respectively, after 3 d. Rapamycin and 3-methyladenine (3-MA) inconsistently activated or inhibited autophagy, respectively, after 3 d. Quantitative PCR (qPCR) for autophagy-related (Atg) genes showed that pattern of expression of lc3 and gabarap best reflected the LC3II signal using WB, while atg7, atg12 and atg4 had similar results. A potential feedback loop resulting in broad up-regulation of Atg genes, most notably becn1, lc3, gabarap and atg9, was seen after treatment with 3-MA and CQ. Autophagy activity in RTgill-W1 was significantly (p < 0.05) decreased for 2 d after infection with viral hemorrhagic septicemia virus (VHSV) IVb. Using qPCR and WB, the VHSV gene copy number and viral N-protein were suppressed by autophagy inhibiting (DON; p < 0.001 and 3-MA; p < 0.05) and blocking (CQ; p < 0.05) chemicals. In an in vivo trial, rainbow trout were experimentally infected with Flavobacterium psychrophilum. Fish fed 5ppm DON (p < 0.001) or pair-fed (p < 0.05) for 12 d before and then during infection had significantly decreased mortality after infection, while the effect of 250ppm CQ was marginally significant (p = 0.052). After 15 d treatment, the LC3II signal was significantly increased in the liver (p < 0.01) and decreased (p < 0.01) in the muscle of fasted fish. The LC3II signal was also significantly increased in the liver of DON-treated fish (p < 0.05) but there were no significant differences between treatments in the spleen. Pair-fed and fasted fish had significantly (p < 0.05) increased expression of gabarap, atg7 and atg12 in muscle after 15 d while 250ppm CQ significantly (p < 0.05) up-regulated hepatic becn1 and atg4. Autophagy modulation alone cannot explain the resistance of rainbow trout to experimental infection with F. psychrophilum. Ministry of Education (Taiwan), Canadian Natural Sciences and Engineering Research Council, Ontario Ministry of Agriculture Food and Rural Affairs, Ontario Ministry of Natural Resources