Background: MicroRNAs participate in many molecular mechanisms and signaling transduction pathways that are associated with plant stress tolerance by repressing expression of their target genes. However, how microRNAs enhance tolerance to low temperature stress in plant cells remains elusive. Objective: In this investigation, we demonstrated that overexpression of the rice microRNA528 (OsmiR528) increases cell viability, growth rate, antioxidants content, ascorbate peroxidase (APOX) activity, and superoxide dismutase (SOD) activity and decreases ion leakage rate and thiobarbituric acid reactive substances (TBARS) under low temperature stress in Arabidopsis (Arabidopsis thaliana), pine (Pinus elliottii), and rice (Oryza sativa). Methods: To investigate the potential mechanism of OsmiR528 in increasing cold stress tolerance, we examined expression of stress-associated MYB transcription factors OsGAMYB-like1, OsMYBS3, OsMYB4, OsMYB3R-2, OsMYB5, OsMYB59, OsMYB30, OsMYB1R, and OsMYB20 in rice cells by qRT-PCR. Results: Our experiments demonstrated that OsmiR528 decreases expression of transcription factor OsMYB30 by targeting a F-box domain containing protein gene (Os06g06050), which is a positive regulator of OsMYB30. In OsmiR528 transgenic rice, reduced OsMYB30 expression results in increased expression of BMY genes OsBMY2, OsBMY6, and OsBMY10. The transcript levels of the OsBMY2, OsBMY6, and OsBMY10 were elevated by OsMYB30 knockdown, but decreased by Os- MYB30 overexpression in OsmiR528 transgenic cell lines, suggesting that OsmiR528 increases low temperature tolerance by modulating expression of stress response-related transcription factor. Conclusion: Our experiments provide novel information in increasing our understanding in molecular mechanisms of microRNAs-associated low temperature tolerance and are valuable in plant molecular breeding from monocotyledonous, dicotyledonous, and gymnosperm plants.