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Goatpox virus (GTPV) is prevalent in goats and is associated with high mortality. This virus causes fever, skin nodules, lesions in the respiratory and lymph node enlargement. Considering the safety risks and side effects of vaccination with attenuated live GPTV vaccine strain AV41, an attenuated goatpox virus (GTPV-TK-ORF), was constructed by deleting non-essential gene fragments without affecting replication and related to the virulence and immunomodulatory functions of the goatpox virus AV41 strain (GTPV-AV41) using homologous recombination and the Cre (Cyclization Recombination Enzyme)/Loxp system. The results of both in vivo and in vitro experiments demonstrated that GTPV-TK-ORF was safer than wild type GTPV-AV41, possessed satisfactory immunogenicity, and could protect goats from a virulent GTPV-AV40 infection. Moreover, the IFN-γ, GTPV-specific antibody, and neutralizing antibody levels in the GTPV-TK-ORF-immunized group were significantly higher than that in the normal saline control group following immunization (P < 0.01). Thus, GTPV-TK-ORF may be used as a potential novel vaccine and viral vector with good safety and immunogenicity.An attenuated goatpox virus (GTPV-TK-ORF) was constructed by homologous recombination and Cre/Loxp system.The deletion of TK gene and ORF8-18 in GPTV-TK-ORF significantly reduced the in vitro virulence of GTPV-AV41.The absence of the deleted gene segments led to a reduction in the virulence of GTPV-TK-ORF in small animals and goats.GTPV-TK-ORF was able to protect goats from a virulent GTPV-AV40 infection.