Rapeseed (Brassica napusL.) is an important crop for edible oil, vegetables, and biofuel. Rapeseed growth and development require a minimum temperature of ~1–3 °C. Notably, frost damage occurs during overwintering, posing a serious threat to the productivity and yield of rapeseed. MYB proteins are important transcription factors (TFs) in plants, and have been proven to be involved in the regulation of stress responses. However, the roles of the MYB TFs in rapeseed under cold stress conditions are yet to be fully elucidated. To better understand the molecular mechanisms of one MYB-like 17 gene,BnaMYBL17, in response to low temperature, the present study found that the transcript level ofBnaMYBL17is induced by cold stress. To characterize the gene’s function, the 591 bp coding sequence (CDS) from rapeseed was isolated and stably transformed into rapeseed. The further functional analysis revealed significant sensitivity inBnaMYBL17overexpression lines (BnaMYBL17-OE) after freezing stress, suggesting its involvement in freezing response. A total of 14,298 differentially expressed genes relative to freezing response were found based on transcriptomic analysis ofBnaMYBL17-OE. Overall, 1321 candidate target genes were identified based on differential expression, including Phospholipases C1 (PLC1), FCS-like zinc finger 8 (FLZ8), and Kinase on the inside (KOIN). The qPCR results confirmed that the expression levels of certain genes showed fold changes ranging from two to six when compared betweenBnaMYBL17-OE and WT lines after exposure to freezing stress. Furthermore, verification indicated thatBnaMYBL17affects the promoter ofBnaPLC1,BnaFLZ8,andBnaKOINgenes. In summary, the results suggest thatBnaMYBL17acts as a transcriptional repressor in regulating certain genes related to growth and development during freezing stress. These findings provide valuable genetic and theoretical targets for molecular breeding to enhance freezing tolerance in rapeseed.