5-Hydroxymethylfurfural causes reproductive toxicity in male mice by increasing oxidative stress and apoptosis through the Nrf2/HO-1 signaling pathway

5-Hydroxymethylfurfural (HMF) is a furan derivative compound commonly found in heat-treated carbohydrate-rich foods. Although its toxicological properties are well-studied, its effects on the male reproductive system at cellular and molecular levels remain unclear. This study is one of the first to evaluate the toxicity of HMF on the male reproductive system by conducting comprehensive analyses in both <i>in vitro</i> and <i>in vivo</i> models. In the <i>in vitro</i> model, TM3 Leydig cells were divided into four groups: control, 0.1, 1, and 10 mM HMF for 24 h. The study evaluated cell cytotoxicity and proliferation, oxidative stress levels, and antioxidant enzyme activity. The mRNA expression levels of oxidative stress- and apoptosis-related genes (<i>Sod1, Gpx1, Nrf2, Ho1, Keap1, Bax, Bcl-2, Casp3, Trp53,</i> and <i>Parp1</i>) were analyzed by RT-qPCR. In the <i>in vivo</i> model, BALB/c mice were divided into three groups: control, 30 mg/kg, and 300 mg/kg HMF administered orally for 21 days, and testicular tissues were evaluated with similar biochemical and genetic analyses. HMF significantly increased cytotoxicity, suppressed proliferation, and caused a significant increase in ROS levels in TM3 cells (<i>p</i> < 0.05). Moreover, HMF increased lipid peroxidation, suppressed antioxidant enzyme activities, and altered the expression of oxidative stress- and apoptosis-related genes in both TM3 cells and testicular tissue (<i>p</i> < 0.05). These statistically significant findings demonstrate that HMF induces oxidative damage and impairs cellular defense and survival mechanisms. In summary, our results highlight the potential reproductive risks associated with dietary HMF exposure and support the need for reassessing its toxicological safety limits.