An interspecific hybrid F1 of Cucumis hystrix Chakr. × Cucumis sativus L. (NC4406) was used to establish the developmental sequence and to characterize the male and female gametophytes at cytological level for further understanding of the phylogenic relationship and the mechanism of fertility or sterility in the interspecific hybrid F1. The development of male and female gametophytes was studied through meiotic analysis and paraffin section observation technique, respectively.Meanwhile, the fertility level was assessed through hybrid F1 backcrossing to cultivated cucumber 4406. Variable chromosome confgurations were observed in the pollen mother cells (PMCs) of hybrid F1 at metaphase Ⅰ, e.g., univalents,bivalents, trivalents, quadravalents, etc. At anaphase Ⅰ and Ⅱ, chromosome lagging and bridges were frequently observed as well, which led to the formation of polyads and only a partial number of microspores could develop into fertile pollen grains (about 23.3%). Observations of the paraffin sections showed numerous degenerated and abnormal embryo sacs during the development of female gametophytes, and only 40% of the female gametophytes could develop into normal eight-nuclear megaspore. On an average, 22.8 and 6.3 seeds per fruit could be obtained from the reciprocal backcross. The interspecific hybrid F1 of C. hystrix × NC4406 was partially fertile; however, the meiotic behaviors of hybrid F1 showed a high level of intergenomic recombination between C. hystrix and C. sativus chromosomes, which indicated that it plays an important role for introgression of useful traits from C. hystrix into C. sativus.