Glycine betaine (GB) treatment is considered effective in preventing low ester production in cold-stored ‘Nanguo’ pears (Pyrus ussuriensis Maxim.). Here, the mechanism of GB on ethylene biosynthesis and signal transduction to accelerate esters accumulation was investigated. We observed a higher ethylene production in GB-treated ‘Nanguo’ pears than that in controls. Genes directly related to ethylene biosynthesis (PuACO1, PuACO2, and PuACS) and ethylene response factors (PuERF2, PuERF109-like, PuERF071-like, PuERF015, PuERF114, PuRAV1-like, and PuERF113-like) were upregulated during cold storage and subsequent shelf at room temperature, and positively contributed to biosynthesis of esters. Moreover, it was found that pears treated with GB increased the content of GB by improving the activity and expression of choline monooxygenase and betaine dehydrogenase, whereas high GB content was positively correlated with high ethylene production. Collectively, these results suggested that triggering of the biosynthesis of ethylene and signal transduction pathways may be the mechanism responsible for the conservation of esters by GB treatment.