Abstract: UL14 is a highly conserved protein among alphaherpesvirus. However, until now, little is known about the function of pseudorabies virus (PRV) UL14. In this study, an UL14-negative PRV mutant (PRV-UL14D) was constructed by inserting an EGFP expression cassette into the UL14 coding sequence. In noncomplementing cells, the mutant was able to replicate, but exhibited an extended growth cycle and significantly reduced plaque sizes, when compared to the field PRV-Ea. These deficiencies were corrected in UL14-expressing cells. In vivo, the average death time of BALB/c mice inoculated with PRV-UL14D was obviously delayed, compared with the same dose of PRV-Ea. From the above results it was therefore concluded that the PRV UL14 product is not essential for virus replication in vitro but delay virus replication and is involved in cell-to-cell spread.