In plant species, the level of polyunsaturated fatty acids (PUFAs) is essential for cold acclimation. To test whether changes in PUFA levels can lead to the alteration of freezing tolerance in poplar trees, we up- and down-regulated a Populus tomentosa Delta-12 fatty acid desaturase gene (PtFAD2) in the hybrid poplar (P. alba x P. glandulosa) clone 84 K. Real-time PCR results demonstrated that compared to untransformed control lines, the transcriptional level of PtFAD2 increased by up to 90% in over-expressing poplar lines (line OE-1) and decreased in down-regulated RNAi lines by up to 64% (line DR-1). As a result, the content of linoleic (C18:2) and linolenic (C18:3) unsaturated fatty acids (FAs) in total FAs increased by 7.5 and 3.9%, respectively, in the OE-1 line and decreased by 14.4 and 5.4% in the DR-2 line when compared to non-transgenic lines. After freezing treatment at -4 degrees C for 3 h without pre-cold acclimation, the survival rates of the PtFAD2-over-expressing cuttings were significantly higher (60% for OE-1) than those of non-transgenic plants (36.7%) and down-regulated lines (10% for DR-2). These results clearly demonstrate that the expression level of PUFAs substantially affected the freezing tolerance of hybrid poplar cuttings and could thus be utilized as an effective strategy to improve poplar anti-freezing traits through genetic engineering biotechnology.