Roses, a popular ornamental crop, often face various abiotic stresses during growth and development, such as cold, drought, and salinity. Rosa multiflora is a commonly used rootstock and exhibits strong resistance to both biotic and abiotic stresses, making it an ideal material for studying mechanisms for resistance. Among the largest plant families, MYB transcription factors play a crucial role in plant abiotic stresses. Our previous research has indicated that RmMYB44 could be involved in the low-temperature response of R. multiflora. This study further investigated RmMYB44, revealing that its expression levels were upregulated in response to chilling, drought, and salt stress. The results suggested its potential role as a key transcription factor in plant resistance to abiotic stresses. Additionally, RmMYB44 encoded a nuclear-localized protein without the self-activating function. The overexpression of RmMYB44 in tobacco plants enhanced the resistance to cold, drought, and salt stresses, as evidenced by the improved growth compared to wild-type (WT) plants under conditions of 4 °C, 30% water-holding capacity, and 200 mM of NaCl, respectively. Moreover, in overexpression tobacco plants, the levels of hydrogen peroxide and malondialdehyde (MDA) were significantly reduced; and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); as well as the proline content and the expression levels of NtPOD, NtCAT, and NtCBF; were significantly elevated under abiotic stresses. We assumed that the resistance to abiotic stress in plants conferred by RmMYB44 was associated with the regulation of cell membrane integrity. This study aimed to elucidate the role of the RmMYB44 gene in the resistance mechanism of R. multiflora against abiotic stress, thereby providing a candidate gene for the molecular breeding of abiotic stress resistance in roses and related species.