Plants have established strategies to improve cold/freezing tolerance through cold acclimation in response to low-temperature stress in winter. After 10 years acclimation in cultivation to the north, the Plumbago auriculata L. has been able to overwinter in field, but the key genes and mechanisms of cold tolerance were not yet clear. MYB transcription factors were widely involved in plant growth and development, metabolism, and abiotic stress regulation. In particular, little has been reported tropical evergreen plants and their regulatory mechanisms in response to cold stress or cold acclimation. The results showed that the short-term artificial cold domestication for 10 d resulted in higher cold hardiness in Plumbago auriculata L., which explained from a physiological level that cold acclimation is an effective way to improve cold resistance. By RNA-seq profiling, PaMYBS3 was identified as a signaling gene that contributes to winter dormancy and inhibits growth and development in Plumbago auriculata L., acting as a negative regulator. Specifically, OE- PaMYBS3 promoted the growth and development compared with the TC- PaMYBS3⁷⁸ and TC- PaMYBS3¹⁵⁹ conserved domain mutant lines in Arabidopsis thaliana, while freezing stress resulted in decreased photosynthetic performance, increased H ₂O ₂, O ^2-, MDA, low soluble sugar content, and weaker cold tolerance. Virus-induced gene-silencing mediated knockdown of PaMYBS3 inhibited the growth and development of Plumbago auriculata L., and the physiological data in VIGS lines after cold stress showed that the low temperature adaptability was improved. In addition, based on RT-qPCR, the expression levels of regulating metabolism related genes in cold acclimation and VIGS lines were verified, including low-temperature dormancy, plant hormones, carbohydrates, etc., which indirectly proved that Plumbago auriculata L. had both CA and WD ecological strategies to resist cold. During cold acclimation, PaMYBS3 promoted ecological dormancy by inhibiting the growth and development cold acclimation, and enhanced cold tolerance in a CBF-independent pathway by maintaining stable carbohydrate metabolism, phytohormone signals, osmoregulatory substances, and ROS metabolism. In summary, this study provides new insights into the way PaMYBS3 responded to low temperatures, and it was regulated in tropical evergreens, using Plumbago auriculata L. as an example. It also provides more evidence for its theoretical basis for overwintering in field.