Abstract: Three plasmids, namely pcDNAE0/2/012, pHIT60 (including the structural genes of MuLV) and pHIT111 (including the retroviral genome, containing LacZ as a reporter) were cotransfected into HEK293T cells for the production of pseudotyped virions with E0/2/012 glycoproteins of classical swine fever virus (CSFV) Shimen strain. The retroviral supernatants were harvested at 48 hours posttransfection and used in western blot and infection assays. Westernblotting revealed only E012 could be expressed on the virions, indicated that the glycoprotein E012 was incorporated onto the retroviral virions. Infection test were performed on SK6, PK15, ST, BHK21, Vero, COS7, HEK293T and CEF cells. The results showed that SK6, PK15 and ST infected were LacZ positive, indicating viral entry, and revealed that the pseudtype virions of MuLVE012 were infectious. To assess whether the CSFV pseudotyped virus entry is pHdependent, PK15 cells were treated with wellcharacterized lysosomotropic agent NH4Cl. Treatment with 30 mmol·L-1 NH4Cl caused ＞90% inhibition of infection by MuLVE012 or MuLVVSV G. These data indicated that the MuLVE012’s entry may be pHdependent. The pseudotyped MuLVE012 particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for CSFV neutralizing antibody. Neutralization titers measured by this assay were highly parallel with those measured by the assay using live CSFV high virulence strain. Because the pseudotype assay does not require handling live CSFV virus, it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.