This study aimed to investigate how low temperature alters the sex expression of monoecious cucumbers (Cucumis sativus L.). Plants were grown under different day/night temperature regimes, 28 °C/18 °C (12 h/12 h), 18 °C/12 °C, 28 °C/12 °C, and 28 °C/(6 h 18 °C+6 h 12 °C). It was found that plant femaleness is highest in the 28 °C/(6 h 18 °C+6 h 12 °C) treatment. Analysis of endogenous phytohormones and sugars in the shoot apex revealed that plant femaleness is positively correlated with the levels of ethylene, abscisic acid (ABA), glucose, and sucrose. Exogenous application experiments suggest that ABA and ethylene biosynthesis, as well as plant femaleness, was enhanced by glucose, sucrose, and mannose, but not by 3-O-methylglucose. Exogenous ABA had no significant effect on ethylene biosynthesis and plant femaleness. Both low temperature- and sugar-induced ABA biosynthesis, ethylene evolution, and plant femaleness can be antagonized by the hexokinase inhibitor glucosamine and the ABA biosynthesis inhibitor nordihydroguaiaretic acid. It is concluded that the enhancement of cucumber femaleness under various temperature regimes is induced by elevated levels of glucose and sucrose in the shoot apex through a sugar signalling pathway involving hexokinase.