Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) displays important functional diversity in mammalian and plants. So far, however, studies on GAPDH have not included the halotolerant, unicellular green alga Dunaliella salina. In the present study, a GAPDH cDNA was cloned and sequenced from D. salina. It was 1394 bp long, with an open reading frame of 1128 bp encoding 376 amino acid residues, and shared a high homology with other organisms. The coding region of the gene was heterologously expressed in E. coli, confirming that the gene cloned from D. salina is indeed GAPDH. Furthermore, the recombinant plasmid p7NBTFIR was constructed to express hairpin RNA (hpRNA) containing sequences homologous to the GAPDH gene to investigate the expression profile of GAPDH by RNAi in D. salina. The results of real-time quantitative PCR revealed that the relative transcription levels of the GAPDH gene in transformants G1 and G2 were reduced to 41.2% and 67.4%, respectively, of the wild-type D. salina. Observations under phase-contrast microscopy showed that the motility of the transformants cell was sluggish. The results of a photoaccumulation experiment showed that the cell motility of transformants G1 and G2 was less active than that of wild-type D. salina. The findings of this study may be useful for further studies on the subcellular localization and functional analysis of the GAPDH gene in microalgae.