The complete sequence of mitochondrial genome of Daphnia magna was determined using long PCR, normal PCR and primers walking approaches. The results showed that the entire mitochondrial genome of Daphnia magna is 14948 bp in size, in which the content of A, T, G, C is 32.37%, 34.73%, 15.58%, 17.31%, respectively. The mitochondrial genome organization and gene order of Daphnia magna were consistent with that of Arthropod, which contains 13 protein-coding genes (PCGs), 22 tRNA genes, 2 ribosomal RNA genes (large and small ribosomal RNAs), and a control region. Except for COI gene using CTG, ATP8 with GTG, and ND3 with ATC, and ND6 with ATT as the initiation codon, and other 9 PCGs of the mtDNA in Daphnia magna started with the typical ATG codon. TAG and TAA were used in 9 PCGs as usual termination codons, except for COI, COII,ND4 and ND5 genes with incomplete termination codon (T). There are 9 intergenic spacer sequencer totaling 81 bp (1-62 bp for each sequence) and 13 overlapping sequences totaling 77 bp (1-27 bp for each sequence), scattered throughout the genome, respectively, and the largest overlap (27 bp) region is located between 16S rRNA and tRNA^Val genes. The secondary structures of 22 tRNAs were predicted and it was found that tRNA^Ser1 failed to form a complete secondary structure, others have typical leaf clover secondary structures. The length of 16S rRNA, 12S rRNA and D-loop were 1373 bp, 752 bp and 289 bp, respectively. The results of this study provide some useful molecular data to clarify the systematical status of Daphnia, as well as its phylogenetic relationships with other species.