Jellyfish venom can cause the mass mortality fish and induces apoptosis of many types of cells; therefore, we speculated that the venom might be able to induces apoptosis in fish. To further understand the mechanism, we cultured Larimichthys crocea with Rhopilema esculentum and performed transcriptome analysis of liver, heart, and kidney of the fish. According to KEGG analysis, we selected certain apoptosis-related pathways, such as the P53 signaling pathway and apoptosis, and analyzed the apoptosis-relatedrelated DEGs in these pathways in the three tissues using quantitative real-time reverse transcription PCR. PIG3, CTSL, CytC, AP1 and Gadd45a were all upregulated in three tissues; whereas Mdm4 was downregulated in heart tissues. Then, we treated HEK 293 T cells with jellyfish venom and knocked down the expression of PIG3 and CTSL using small interfering RNAs (siRNAs). In cells treated with jellyfish venom, cell viability increased significantly after RNAi; P53 and CASP9 were upregulated, but the expression of CASP9 was much higher in the Control-RNAi group (without siRNA). PIG3 and BAX were downregulated in the PIG3-RNA+ (with PIG3 siRNA); CTSL and CYTC were upregulated, but BCL2 was downregulated in the Control-RNAi group. The protein levels of PIG3, CTSL, and CASP9 detected by western blotting showed similar trends to their mRNA expression levels. Thus, the results indicated that jellyfish venom could activate PIGs through the P53-PIG3 pathway to damage lysosomes, which released CTSL to affect mitochondria, leading to the apoptosis in fish. The data increases our understanding of the mechanism of apoptosis induced by jellyfish venom. Display omitted •Jellyfish venom can cause mortality of fish and apoptosis of many model cells.•Apoptosis relating genes were highly expressed in fish treated by jellyfish venom.•RNAi depressed the expression of Cyt C and Casp9 in cell treated by jellyfish venom.•Jellyfish venom could induced apoptosis of fish by PIG3 of P53-PIG3-CTSL pathway.•The data increases understanding of mechanism for cytotoxicity of jellyfish venom.