The ploidy levels affect the physiological traits and stress tolerance in plants. In comparison to diploids, tetraploids enable to own advanced physiological traits and increase the size and fruit quality and with resistance to abiotic stresses. However, Ziziphus jujuba Mill. ‘Dongzao’ is one of the most popular and consumer favorite fresh fruit in China, which exhibits cold sensitivity within field observation when it is autotetraploid in compared to diploid. Thus, we investigated the molecular mechanism of differential cold tolerance between autotetraploids and diploids in this study. The morphological and physiological analysis under 4 °C treatment conformed that diploids were cold tolerant than autotetraploids. The comparative transcriptome analysis indicated that the diploid samples expressed 56 and 694 differential expressed genes (DEGs) at 6 h and 24 h after cold exposure, while the number of DEGs increased in autotetraploid samples at 6 h (225) and 24 h (2774). According to Go annotation analysis, DEGs were classified into different functional groups including catalytic activity, binding and transporter activity, metabolic pathways, cellular process and response to stimulus. Furthermore, we performed genes clustering and GO enrichment analysis regarding to 882 DEGs and observed the involvement of cold responsive genes in the xyloglucan metabolite process, calcium ion transmembrane transport, and microtubule-based movement/process. Among these DEGs, three genes including Tub1, CML41, and IQD1 showed a significantly differential expressing pattern between diploids and autotetraploids and could be regulated by microtubules changes. These results indicated that microtubules play a crucial role in the expression of CML41 and IQD1 to regulate the cold resistance of diploids and provide the theoretical foundation for the polyploid breeding of cold resistance in Chinese jujube.