This study aimed to investigate the interactive effects between selenium and vitamin B6 supplementation in the transsulfuration pathway and glutathione metabolism of juvenile rainbow trout (Oncorhynchus mykiss) and in response to oxidative stress. Four plant protein-based diets (48% crude protein and 23% total lipid) naturally low in selenium and vitamin B6 were designed: CTL, without any selenium or vitamin B6 supplementation; SEL, supplemented with 4 mg selenium per kg diet supplied as selenomethionine (SeMet); PYR, supplemented with 50 mg pyridoxine hydrochloride (PN.HCL) per kg diet and SEPY, co-supplemented with SeMet and PN.HCL. Groups of 50 juvenile rainbow trout (28 ± 3 g) were randomly distributed in a flow-through system in triplicate tanks per treatment and fed on one of the experimental diets two times per day for twelve weeks. In addition, 15 fish per tank were exposed to periodic hyperoxia stress for one week prior to sampling. Therefore, the dissolved oxygen levels in the tanks were increased from 8 mg/l to 13 mg/l during 8 h per day. Dietary SeMet supplementation increased feed intake (1.44 ± 0.03 vs. 1.49 ± 0.02), but without any significant differences in final body weight (180 ± 3 vs 184 ± 3 g) between groups. SeMet supplementation was associated with increased liver and muscle S-adenosylhomocysteine levels. There was a general decrease of transsulfuration metabolites by SeMet supplementation in muscle tissue. On the other hand, PN.HCL supplementation increased the gene expression of the first step transsulfuration pathway enzyme, cbs, in liver tissue with similarly higher levels of transsulfuration metabolite homocysteine and total glutathione. Fish subjected to periodic hyperoxia showed lower reduced glutathione levels in liver tissue, which indicates modifications to the cellular redox system of fish in response to the stress. In stressed fish, interactive effects of SeMet and PN.HCL supplementation were detected on transsulfuration metabolites in both liver and muscle tissue. The results show that SeMet and PN.HCL supplementation can induce contrasting effects in the transsulfuration system of fish. The significant interactions in stressed fish between SeMet and PN.HCL supplementation indicate that both nutrients are required to maintain glutathione homeostasis under challenging environmental conditions.