Polyunsaturated fatty acids (PUFAs) are essential for mammalian testis development and sperm function. However, PUFAs that are contained in linseed oil are easily oxidized in the diet and biohydrogenated in the rumen. In this study, we investigated the effect of linseed as a source of PUFAs on the antioxidant capacity and testis development in Hu lamb. Seventy-five 3-month-old lambs were randomly assigned to three groups. Within each treatment group, 25 lambs were allocated to five pens (five lambs per pen). The lambs in the control group were fed a control diet without linseed for 42 days from D22 to D63. Group I (BS28) was fed a control diet from D22 to D35 and 8% linseed diet from D36 to D63. Group II (BS42) was fed an 8% linseed diet for 42 days from D22 to D63. After 63-day feeding trial, all lambs except the heaviest and lightest in each pen were humanely slaughtered and investigated. Results revealed that feeding linseed did not affect the body weight, scrotal circumference, and testis weight, whereas feeding linseed for 42 days increased the epididymis weight (37.85 ± 1.61 g vs. 32.09 ± 1.06 g, P < 0.05) compared with the control group. Feeding lambs with linseed for 42 days also significantly upregulated the expression of antioxidative (glutathione peroxidase 4 and copper-zinc superoxide dismutase), steroidogenesis (3β-hydroxysteroid dehydrogenase and steroid acute regulatory protein), and PUFA metabolism-related genes (fatty acid desaturase 2 and elongation of very long-chain fatty acid protein 2) and proliferating cell nuclear antigen mRNA (P < 0.05). It also increased the relative expression of mitochondrial DNA (P < 0.05), total antioxidant capacity (0.230 ± 0.019 mmol/mgprot vs. 0.175 ± 0.011 mmol/mgprot, P < 0.05), and superoxide dismutase (1661.467 ± 147.117 U/mgprot vs. 1158.891 ± 98.850 U/mgprot, P < 0.05) in testicular tissue but decreased the cholesterol concentration (0.331 ± 0.073 mmol/mgprot vs. 0.671 ± 0.092 mmol/mgprot, P < 0.05) compared with the control group. Therefore, feeding lambs with linseed for 42 days stimulated seminiferous tubule development and increased the number of Sertoli cells (20.71 ± 0.89 vs. 17.6 ± 0.73, P < 0.05), epididymal cauda lumina diameter (638.26 ± 22.32 μm vs. 444.41 ± 34.80 μm, P < 0.05), and the number of sperm in the epididymal cauda (68.91 ± 7.06 × 10/g vs. 36.61 ± 7.50 × 10/g). All these results suggested that feeding linseed in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and PUFA metabolism-related genes; increased the antioxidant capacity in lamb's testis; and contributed to testis development and spermatogenesis.Copyright © 2020 Elsevier Inc. All rights reserved.