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HighlightsThe model of S. eriocheiris infection 3T6 cells was established.A new method to assay infection rates, S. eriocheiris‐oxytetracycline protection assays, was established.The 3T6 cells global mRNA and miRNA expression profiling were detected after S. eriocheiris infection.Integrated analysis mRNA and miRNA expression profiling after S. eriocheiris infection. &NA; Spiroplasma eriocheiris is a novel pathogen similar to the Spiroplasma mirum and also had an ability to infect the newborn mice and caused cataract. Our study was designed to study how S. eriocheiris infects mouse 3T6‐Swiss albino cells and to elucidate the cellular molecular pathogenesis of Spiroplasma. FCM analysis and MTT analysis clearly shown that S. eriocheiris could induce 3T6 cell apoptosis and cause cell viability decreased seriously. Immunofluorescence experiments and TEM analysis shown that S. eriocheiris can invade 3T6 cells and form typical inclusion bodies and exhibit vacuolization in vitro. S. eriocheiris‐oxytetracycline protection assay show that the infective bacteria already were detected at 1 h post infection, and sharply increased at 12 h after the bacteria infection. To further study the infection mechanism of S. eriocheiris, global mRNA and microRNA (miRNA) expression profiling were analyzed after the cells infected with the bacteria. A total of 619 non‐redundant annotated transcripts (183 up‐regulated and 436 down‐regulated) and 22 miRNAs (8 up‐regulated and 14 down‐regulated) were differential expression after 6 h S. eriocheiris infection compared to control group. Integrated analysis shown that homologous genes from differential expression miRNA targets and the differential expression genes of the mRNA microarray were major focused on two important pathways focal adhesion and MAPK signaling pathway. To validate the results of microarray, eight focal adhesion (&bgr;‐Catenin, Parvin, Grb2 and ERK) and MAPK signaling pathway (FGFR, Grb2, ERK, MKK3, p38 and JNK) genes and the housekeeping gene GAPDH were assayed by qPCR and Western blot to confirm the results. Eight miRNAs (miR‐143‐3p, miR‐214‐5p, miR‐322‐3p, miR‐328‐5p, miR‐351‐5p, miR‐466h‐5p, miR‐503‐5p and miR‐30c‐1‐3p) and the housekeeping gene U6 miRNA were assayed by qPCR to confirm the results of microarray. All the results help us better understand the infection mechanism of S. eriocheiris.