Existing data indicate that YB-1, a gene member of the cold chock domain family, is regulated by cold stress in many species. However, information regarding YB-1 in fish is limited. To gain more insight into the potential biological function of YB-1 after cold stress, we have cloned YB-1 of the spotted sea bass (Lateolabrax maculatus) and studied the expression at transcriptional level of the LmYB-1 gene under low temperature. The cloned LmYB-1 cDNA (1454 bp) includes a 900-bp open-reading frame that encodes a 300-residue protein and contains C-terminal, CSD, and N-terminal domains. The results of real-time quantitative PCR showed that LmYB-1 was expressed in all tested tissues, with the highest expression in liver. Within liver, brain, and heart, LmYB-1 mRNA expression increased during cold stressed fish, compared to control fish. No significant induces in the expression of LmYB-1 were recorded in muscle, except at 10 °C during 24 h cold stress. Overexpression of LmYB-1 in CIK cells caused the upregulation of the expression of Hsc70, SOD1, and SOD2. Our results provided new insights into the LmYB-1 expression during cold stress and warranted further investigation into this potentially important protein in improving fish farming at low temperature.