V-ATPase plays an important role in biological response to various environmental stress, in order to explore the role of V-ATPase in the low temperature adaptation of Pinctada fucata martensii, the V-ATPase-d gene of P. fucata.martensii was identified, and the changes in the expression of Pm-V-ATPase-d under low temperature stress were analyzed. Screening and comparative analysis of the gene in the low temperature resistant breeding line (R) F₃ and Beibu Gulf wild population (W) SNP loci in the exon region. Sequence analysis showed that Pm-V-ATPase-d has a total length of 1 473 bp and open reading frame (ORF) of 1 140 bp, encoding 379 amino acids, with a typical ATP domain, Pfam vATP-synt_AC39. The results of motif analysis and tertiary structure prediction showed that Pm-V-ATPase-d was highly conserved, and it clustered with Crassostrea gigas in the phylogenetic tree. Whole tissue fluorescence quantitative results showed that Pm-V-ATPase-d was expressed in all the tested tissues, and the expression level was higher in hepatopancreas, gonads and gill tissues (P<0.05). Under low temperature stress, the expression level of Pm-V-ATPase-d gene increased first and then decreased with the extension of time, and the expression level of the low temperature group was significantly higher than the control group(P<0.05), indicating that Pm-V-ATPase-d may be involved in the response of P. fucata martensii to temperature stress. The SNP analysis of the exon region of Pm-V-ATPase-d resulted in a total of 35 SNPs, which 34 were synonymous mutations, only one site was a non-synonymous mutation, and 26 SNPs were significantly different between genotypes and alleles in W and R populations(P<0.05), the results of haplotype linkage disequilibrium analysis showed that the Pm-V-ATPase-d gene SNPs could form 6 haplotype blocks and 14 haplotypes, the haplotypes GAAT, CGC, TC, TG and AG were significantly correlated with low temperature resistance traits of P. fucata martensii (P<0.05). These research data indicate that Pm-V-ATPase-d may be a candidate gene involved in regulating the adaptation to low temperature of P. fucata martensii. This study can provide a research basis for the adaptation mechanism of P. fucata martensii to low temperature. The SNPs and haplotypes associated with low temperature resistance traits can be used in molecular assisted breeding.