The cucumber (Cucumis sativusL.) is an important vegetable crop worldwide, and fruit trichomes or spines are an important trait for external fruit quality. The mechanisms underlying spine formation are not well understood, but the plant-specific NAC family of transcription factors may play important roles in fruit spine initiation and development. In this study, we conducted a genome-wide survey and identified 91 NACgene homologs in the cucumber genome. Clustering analysis classified these genes into six subfamilies; each contained a varying number of NACfamily members with a similar intron–exon structure and conserved motifs. Quantitative real-time PCR analysis revealed tissue-specific expression patterns of these genes, including 10 and 12 that exhibited preferential expression in the stem and fruit, respectively. Thirteen of the 91 NACgenes showed higher expression in the wild-type plant than in its near-isogenic trichome mutant, suggesting their important roles in fruit spine development. Exogenous application of four plant hormones promoted spine formation and increased spine density on the cucumber fruits; several NACgenes showed differential expression over time in response to phytohormone treatments on cucumber fruit, implying their essential roles in fruit-trichome development. Among the NACgenes identified, 12 were found to be targets of 13 known cucumber micro-RNAs. Collectively, these findings provide a useful resource for further analysis of the interactions between NACgenes and genes underlying trichome organogenesis and development during fruit spine development in cucumber. A group of genes connected with spine growth in cucumber fruits could enable the development of cultivars for specific markets. In China, people prefer cucumbers with plentiful spines, while for the European market, cucumbers are usually smooth. This has implications for growers, yet the molecular mechanisms inherent in spine or 'trichome' growth are not completely clear. Yiqun Weng at the University of Wisconsin-Madison in the United States, together with Huazhong Ren at the China Agricultural University in Beijing, and co-workers identified genes related to trichomes in cucumbers. In a genome-wide survey, the researchers identified 91 genes from a family of transcription factors predicted to influence trichome growth. Of these, six genes were highly expressed in wild-type spiny cucumbers, while a further three were found highly expressed in mutant smooth cucumbers, indicating that they may reduce spine growth.