Abstract: 【Objective】To explore the codon bias of the mitochondrial genome in Sipunculus nudus for the effects of natural selection and genetic mutation on the codon bias, and make full use of the characteristics of the bias to provide scientific basis for the molecular genetic improvement of S.nudus. 【Method】According to the mitochondrial genome sequence of S.nudus, ten non-repetitive gene sequences with more than 300 bp in length and starting with ATG were selected as research objects. CondonW 1.4.2 was used to analyze the codon bias parameters of the mitochondrial genome in S.nudus.Neutral plot analysis, ENC-plot analysis, PR2-plot analysis and corresponding analysis were applied to reveal the dominant factors in the formation of codon bias of the mitochondrial genome, and optimal codons in the mitochondrial genome were determined based on high-frequency and high-expression codons. 【Result】The GC content of the mitochondrial gene codons varied with the position. The averages of GC1, GC2 and GC3 were 51.06%, 40.19% and 47.11% respectively.The average GC content was 46.12%; the averagesof ENC, CAI, and CBI were 50.022, 0.150 and-0.023 respectively; there were 31 synonymous codons of RSCU>1.00 with codons ending with A or C accounting for 87.10%. The neutral plot analysis showed that there was no correlation between GC12 and GC3, with the correlation coefficient being 0.05; the ENC-plot analysis indicated that all tested genes were located below the standard curve, suggesting that the codon bias of the mitochondrial genome was shaped by natural selection; the PR2-plot analysis showed that nine genes were scattered in the second quadrant of the coordinate system, except ND6 gene on the left x-axis; the corresponding analysis showed that the contribution rates of the first four axes were 23.86%, 14.96%, 12.21% and 10.82% respectively. In the coordinate system, the distribution of cytochrome C genes was relatively concentrated, while the distribution of NADH reductase genes was relatively scattered. Based on the high-frequency and high-expression codons in the mitochondrial genome, nine codons including GUA, CUC, CAC, CUU, AUU, CCU, GCA, AAC and GAA were finally determined to be the optimal codons in the mitochondrial genome in S. nudus. 【Conclusion】The codon bias of the mitochondrial gene of S. nudus is weak, preferring codons ending in A/C; natural selection plays a dominant role in shaping the codon bias of the mitochondrial genome in S.nudus. Therefore, exogenous genes can be optimized on the basis of the codons bias of mitochondrial gene in the host to improve their expression efficiency and accelerate the genetic improvement of varieties of S. nudus.