MicroRNA394 (miR394) and its target gene LEAF CURLING RESPONSIVENESS (LCR) are known to mediate plant growth and development. However, whether miR394 and LCR regulate plant adaptation to low temperature has not been investigated. Here, we showed that miR394a overexpressing (35S::MIR394a) plants and a LCR loss of function lcr mutant exhibited more tolerance to cold stress (4 to −11°C), whereas LCR overexpressing (35S::m5LCR) plants displayed a cold hypersensitive phenotype as compared to wild-type. Both 35S::MIR394a and lcr plants accumulated more proline and total soluble sugars under cold stress (4°C) than wild-type. Furthermore, a lower rate of electrolyte leakage was observed in 35S::MIR394a and lcr plants. These results indicated that the physiological response was changed due to the expression of miR394. However, a contrasting phenotype was found in LCR overexpressing plants. Analysis of the cold-responsive genes revealed that transcripts of CBF1, CBF2 and CBF3 (C-repeat/dehydration binding factor) were increased in the 35S::MIR394a plants relative to the wild-type, suggesting that miR394-regulated cold tolerance was involved in the CBF-dependent cold responsive pathway. Taken together, our results showed that both miR394 and its target gene LCR are involved in plant response to low temperature.