Strawberry ( Fragaria × ananassa Duch.) is sensitive to salt stress. Application of exogenous 5-aminolevulinic acid (ALA) can induce chloride channel ( CLC) gene expression, which promotes Cl - interception in roots and reduces transport to shoots, thereby improving plant salt tolerance. However, the mechanism of ALA-induced transcriptional regulation of FaCLC still remains unknown. In this paper, 23 FaCLC genes were identified in the whole genome of strawberry, which can be classified into two subclasses and six subgroups. NaCl stress stimulated the expression of FaCLC-b1/c4/e3 in the leaves and roots of strawberry, and ALA further promoted the gene expression under salt stress. NaCl and ALA activated the transcriptional activity of three gene promoters. Subcellular localization analysis showed that FaCLC-b1 and FaCLC-c4 are tonoplast proteins. Overexpression of FaCLC-b1 and FaCLC-c4 in tobaccos improved the salt tolerance of transgenic plants, with more Cl - retention in the roots and less accumulation in the leaves. Furthermore, we identified a nucleus-localized transcription factor FaMYB44. Verification by Y1H, LUC, and EMSA demonstrated that FaMYB44 can recognize the MBS element of the promoter of FaCLC-c4 and negatively regulate its expression. NaCl stress induced FaMYB44 expression in strawberry roots, while ALA suppressed its expression. Overexpression of FaMYB44 in tobacco resulted in more Cl - accumulation in the leaves, which reduced salt tolerance of the plant. Thus, FaMYB44 is a negative transcription factor for salt tolerance of strawberry, whose expression is induced by salt stress but downregulated by ALA. FaMYB44 was able to bind to the promoter of FaCLC-c4 to depress its expression, while ALA inhibited FaMYB44 expression, thereby alleviating the suppression of FaMYB44 on FaCLC-c4 expression, and then intercepting Cl - in roots and increasing salt tolerance. These findings provide a new perspective on the transcription regulation of FaCLC genes and facilitate better application of exogenous ALA in salt tolerance practices for fruit production.