The chromosomal gene encoding the phospholipase D from Corynebacterium pseudotuberculosis (biovar ovis) isolate Whetten 1 was replaced with an allele containing a nonsense mutation. The virulence of the mutant strain (W1.31r1) and the isogenic parental strain were then compared by inoculation of goats. The wild-type strain caused abscessation at the site of infection, which then spread to the regional lymph node, while W1.31r1 had a reduced ability to establish a primary infection and was incapable of dissemination. Our results confirm that phospholipase D is a virulence determinant of C. pseudotuberculosis that increases the persistence and spread of the bacteria within the host.