This study identified a new male-sterile germplasm of Chinese jujube, named male-sterile No. 2 (JMS2), and achieved controlled hybridization using that germplasm as the female parent. The anthers of JMS2 before flower bud opening became shrunken, dingy yellow and much smaller than normal ones, and they changed to brown after anthesis. No pollen was observed in anthers of JMS2 and its male-sterile trait remained stable over different years. A total of 1,642 fruits were obtained from ten intra- and interspecific cross combinations via controlled hybridization from 2008 to 2012 using JMS2 as the female parent. Of those, 27.3% produced seeds, on average (0-72.6%). The rate of fruit with seed (RFS) was significantly different between cross combinations or years. Compared to other cross combinations, the RFS in the combination of JMS2 × ‘Xingguang’ (a Chinese jujube cultivar with high resistance to jujube witches’ broom disease) and JMS2 × ‘Xing16’ (a sour jujube genotype) remained high in different years and reached means of 48.7 and 58.1%, respectively. Finally, 150 plantlets were regenerated from immature embryos, and 51 of them were randomly selected and identified to be authentic hybrids using amplified fragment length polymorphism (AFLP) markers. This is the first report of hybrids obtained from a cross between Chinese jujube and sour jujube.