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Chemical investigation of the crude extract of the aerial part of <i>Zygophyllum album</i> L. (<i>Z. album</i>) led to the isolation of a new saponin, Zygo-albuside <b>A</b> (<b>7</b>), together with seven known compounds, one of them (caffeic acid, compound <b>4</b>) is reported in the genus for the first time. NMR (1D and 2D) and mass spectrometric analysis, including high-resolution mass spectrometry (HRMS), were utilized to set up the chemical structures of these compounds. The present biological study aimed to investigate the protective antioxidant, anti-inflammatory, and antiapoptotic activities of the crude extract from the aerial part of <i>Z. album</i> and two of its isolated compounds, rutin and the new saponin zygo-albuside <b>A</b>, against methotrexate (MTX)-induced testicular injury, considering the role of miRNA-29a. In all groups except for the normal control group, which received a mixture of distilled water and DMSO (2:1) as vehicle orally every day for ten days, testicular damage was induced on the fifth day by intraperitoneal administration of MTX at a single dose of 20 mg/kg. Histopathological examination showed that pre-treatment with the crude extract of <i>Z. album</i>, zygo-albuside <b>A</b>, or rutin reversed the testicular damage induced by MTX. In addition, biochemical analysis in the protected groups showed a decrease in malondialdehyde (MDA), interleukin-6 (IL-6) and IL-1β, Bcl-2-associated-protein (Bax), and an increase in B-cell lymphoma 2 (Bcl-2) protein, catalase (CAT), superoxide dismutase (SOD) in the testis, along with an increase in serum testosterone levels compared with the unprotected (positive control) group. The mRNA expression levels of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), p53, and miRNA-29a were downregulated in the testicular tissues of the protected groups compared with the unprotected group. In conclusion, the study provides sufficient evidence that <i>Z. album</i> extract, and its isolated compounds, zygo-albuside <b>A</b> and rutin, could alleviate testicular damage caused by the chemotherapeutic agent MTX.