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Background: Classical swine fever (CSF) caused by CSF virus (CSFV) is highly contagious andcauses significant economic losses in the pig industry throughout the world. Previously we demonstrated that porcine Mx1 (poMx1), when fused to HIV Tat protein transduction domain (PTD), inhibits CSFV propagation in PK-15 cells, but it is unknown whether PTD-poMx1 exhibits antiviral activity in other porcine lines and it is efficacious for controlling CSFV infection in pigs in China., Methods: Two porcine cell lines, ST and 3D4/21, were used to investigate in vitro antiviral activity of PTD-poMx1 against CSFV using confocal microscopy, western blot, flow cytometry, and real-time RT-PCR. Furthermore, in vivo antiviral activity of PTD-poMx1 was assessed by means of rectal temperature, clinical score, pathological lesion, white blood cell count, viral load, etc., Results: PTD-poMx1 entered both cell lines within 3 h and maintained for 16 h, but did not affect CSFV binding and uptake. Viral titers and qRT-PCR data showed that PTD-poMx1 inhibited CSFV replication in both cell lines, showing significant antiviral activity after infection. Injection of PTD-poMx1 into CSFV-challenged pigs attenuated CSFV symptoms and viremia in dose-dependent manner but did not completely block virus replication within 14 days post challenge, suggesting that PTD-poMx1 confers partial protection against a lethal challenge., Conclusion: We demonstrated the anti-CSFV activity of PTD-poMx1 in vitro and in vivo. The results have shown that treatment with PTD-poMx1 alleviated symptoms and viral load in infected pigs. The results support our previous in vitro studies and suggest that PTD-poMx1 could be promising in reducing the clinical signs caused by CSFV.