The current study was aimed to evaluate the protective effect of Holothurian atra (HA) extract; naturally occurring marine resource, against methotrexate (MTX) in- duced testicular dysfunction. Mature rats received either MTX (20 mg/kg, intraperi- toneally) or saline on the 7th day of experiment al design. Seven days prior and after MTX-injection, rats received HA at dose of 300 mg/kg intragastrically (HA + MTX group; HA group alone). Serum was extracted and testicular tissues were examined for the changes in serum biochemistry (liver & kidney biomarkers, testicular hor- mones and antioxidants), molecular and histopthological alterations using RT-PCR and immunohistochemistry. MTX-injected rats induced alteration in all testicular pa- rameters. Prior administration of HA ameliorated the MTX-induced oxidative stress. HA administration normalised MTX-induced decrease in serum levels of interleukin- 6 (IL-6), tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), reproduc- tive hormones (FSH, LH and testosterone) and antioxidants GST, SOD and catalase. MTX-injected rats down-regulated mRNA expression of GST, SOD, steroidogenesis associated genes, IFN-γ, Bcl2 and NFKB. MTX up-regulated BAX expression and cas- pase 9 immunoreactivity that were ameliorated in HA + MTX group. Collectively, HA ameliorated and restored all altered genes. In conclusion, HA is a promising supple- ment that is helpful in protection against testicular cytotoxicity and dysfunction in- duced by methotrexate.