Ethyl methanesulfonate (EMS) is a stable and effective chemical mutagen. In this study, cucumber (Cucumis sativus L. cv. “Shannong No. 5”) seeds were treated by 1% EMS for 12 h, 24 h and 48 h to optimize EMS mutagenesis and determined median lethal dose of EMS (1% EMS and 24 h) for “Shannong No. 5”. After treated by 1% EMS for 24 h, 541 M1 plants were grown in greenhouse for phenotype investigation. The fertility of M1 cucumbers was very low, and only 79 lines produced seeds after self crossing. 60 independent M2 families comprising 600 M2 plants were investigated for phenotypic alteration, and 11 individual mutant lines were isolated into six groups: short-fruit mutants, long-fruit mutants, small-flower mutants, big-flower mutants, opposite-tendril mutants and clustered-leaf mutants. The mutation frequency was 18.3%. Two selected representatives, short-fruit mutants and clustered-leaf mutants, showed 1:3 of segregation ratio in M2 populations. This ratio is consistent with classic Mendelian model, indicating that the two kinds of mutants may be controlled by a single recessive gene, respectively. Long-fruit phenotype was stably inherited and no segregation was observed in M3 generation, indicating that this mutant line may be homozygous.