The research of the synergistic effect of Spd and IAA as well as the effects of temperature difference round the clock, N concentration and pH on the induction of female flowers in cotyledons of cucumber(Cucumis sativus L.) in vitro was carried out, in which MS basal medium supplemented with Spd and IAA was used. It was found that the induction rate and the amount of female flower in every trial were considerably low and even down to zero when Spd or IAA was respectively added to cotyledons cultured on MS basal medium, while the induction effect were obviously higher when 12 mg·L-1 Spd and 0.01 mg·L-1 IAA were added simultaneously, and no female flowers could be observed on the control medium, which indicated that Spd and IAA had an significant synergistic effect on the induction of female flowers. Experiment was done with N concentration of 60~90 mmol·L-1 or with temperature difference round the clock of 0~10℃, and with pH of 5.4~6.6, the great effect on the induction of female flowers was achieved on medium of N 70 mmol·L-1, from temperature difference 6℃ and pH 6.2, which showed that the appropriate increase of temperature difference, N concentration and pH was of benefit to the induction of female flowers in cotyledons of cucumber(Cucumis sativus L.)