Background and aims <p>Cadmium (Cd) is a prevalent heavy metal pollutant worldwide. Cucumber (<i>Cucumis sativus</i> L.), a globally important vegetable, has high potential to accumulate Cd and is severely affected by its toxicity. However, the variation and potential mechanism of Cd tolerance in cucumber remain poorly understood to date. This study aims to assess the Cd tolerance among different cucumber cultivars and dissect the potential mechanism for Cd tolerance in cucumber.</p> Methods <p>The Cd tolerance of 36 cucumber cultivars was assessed through hydroponic experiments, and the relationships of Cd concentration and accumulation in the roots and shoots and translocation factor with Cd tolerance were analyzed. The expression levels of homologous genes involved in Cd detoxification were quantified in the selected Cd tolerant cultivar and sensitive cultivar. Based on the gene expression levels, <i>CsNramp5</i> (encoding a promising Cd transporter) was identified, and its Cd transport activity was tested in yeast.</p> Results <p>The 36 cucumber cultivars showed significant differences in Cd tolerance. The Cd tolerance was negatively correlated (<i>P</i> &lt; 0.05, r = -0.389) with root Cd concentration, while positively correlated (<i>P</i> &lt; 0.05, r = 0.388) with root Cd accumulation. Moreover, <i>CsNramp5</i> had a higher relative expression level than other genes, and CsNramp5 showed a strong Cd transport activity in yeast.</p> Conclusion <p>The variations of Cd tolerance among 36 cucumber cultivars are primarily driven by the differences in Cd concentration and accumulation in roots, and partly related to Cd uptake regulated by the expression of <i>CsNramp5</i>, which encodes a Cd transporter.</p>