Abstract: Portunus trituberculatus as a popular table delicacy is one of the most important fishery and aquaculture species of crab around the coast of China. In crustaceans, molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ-sinus gland (XO-SG) of the eyestalks, had been proposed to regulate molting by inhibiting the synthesis of ecdysteroids from Y-organs (YO). The method for determining the levels of MIH mRNA in the swimming crab had been developed using relative quantification of quantitative real-time PCR (qRT-PCR). We found the expression level of MIH mRNA was the highest in the XO-SG. By taking surstage D0 as the control group, the levels of MIH mRNA were analyzed by 2-DDCt in a molt cycle, and the results showed that MIH transcripts down-regulated 0.42?.08, increased (1.09?.09, increased 1.35?.16 fold in stage A, B, C, respectively, and down-regulated 0.78?.07, down-regulated 0.27?.08, down-regulated 0.20?.04 fold in surstage D1, D2, D3/4, respectively. In addition, we used the method of high performance liquid chromatography-e1ectrospray ionization tandem mass spectrometry (LC-MS/MS) to complete the process of measuring the consistency of portunus molting ecdysteroid (20-hydroxyecdysone, 20E) in hemolymph. The results showed that the consistency of ecdysone was below the instrument detection limit of 0.33 pg in the post molt stage (A/B). In the inter-molt period (C), the consistency of ecdysone gradually returned to (1.666?.762) ng/mL. In the pre-molt ecdysteroid titer increased gradually to (4.047?.5133), (6.756?.928) and (8.609?.827) ng/mL in surstage D0, D1 and D2, respectively. The ecdysteroid titer increased steadily to a peak of (19.534?.799) ng/mL in the surstage D3, then dropped to 11.616 ng/mL in surstage D4. These stage-specific expression changes in MIH mRNA levels were accompanied by significant fluctuations in hemolymph ecdysteroid titer. During a molt cycle of the swimming crab, the expression of MIH exhibited a negative correlation with ecdysone in hemolymph, and this correlationindicated that MIH regulated the occurrence and advance of molt by inhibiting the ecdysteroid synthesis from YO.