Cucumber is one of the most important vegetables, and nitrogen is essential for the growth and fruit production of cucumbers. It is crucial to develop cultivars with nitrogen limitation tolerance or high nitrogen efficiency for green and efficient development in cucumber industry. To reveal the genetic basis of cucumber response to nitrogen starvation, a genome-wide association study (GWAS) was conducted on a collection of a genetically diverse population of cucumber (Cucumis sativus L.) comprising 88 inbred and DH accessions including the North China type, the Eurasian type, the Japanese and South China type mixed subtype, and the South China type subtype. Phenotypic evaluation of six traits under control (14 mM) and treatment (3.5 mM) N conditions depicted the presence of broad natural variation in the studied population. The GWAS results showed that there were significant differences in the population for nitrogen limitation treatment. Nine significant loci were identified corresponding to six LD blocks, three of which overlapped. Sixteen genes were selected by GO annotation associated with nitrogen. Five low-nitrogen stress tolerance genes were finally identified by gene haplotype analysis: CsaV3_3G003630 (CsNRPD1), CsaV3_3G002970 (CsNRT1.1), CsaV3_4G030260 (CsSnRK2.5), CsaV3_4G026940, and CsaV3_3G011820 (CsNPF5.2). Taken together, the experimental data and identification of candidate genes presented in this study offer valuable insights and serve as a useful reference for the genetic enhancement of nitrogen limitation tolerance in cucumbers.