A new biotype 2 of Y. ruckeri O1, which lacks motility has proven highly virulent for rainbow trout, and is causing disease in cultured trout even in fish vaccinated with commercial ERM biotype 1 vaccines. Not much is known about immunity against biotype 2, and therefore have we produced a Y. ruckeri O1 biotype 2 immersion vaccine and tested the protection against both Y. ruckeri biotype 1 and 2 infections. Seven months post vaccination, both vaccinated and mock-vaccinated groups of rainbow trout were bath challenged with Y. ruckeri serotype O1, biotype 1 or 2. Challenge with biotype 2 resulted in very low mortalities with no significant difference in mortality between vaccinated and mock-vaccinated fish. Challenge with biotype 1 resulted in a significantly lower mortality (P=0.0001) in the vaccinated group. This result was confirmed 15 months post vaccination (P<0.05). Three and seven days post challenge five fish from each group were sampled for RT-qPCR as well as immunohistochemical analysis in order to find increases in molecular markers which correlate with the increased protection in the vaccinated fish. In general the mock-vaccinated group was more infected in all examined organs (head kidney, spleen, liver, brain, muscle, heart, intestine, skin and gill). Seven days post infection 40% of mock-vaccinated fish were still heavy infected, which corresponds well with overall mortality in this group (35%). In general pro-inflammatory cytokine expression was higher in the mock-vaccinated group and correlated to the higher load of Y. ruckeri in the tissues. Some CD markers such as CD4 and CD86 were significantly increased at the mRNA level in the vaccinated fish during challenge, probably reflecting fast induction of adaptive immunity. IgT transcripts were also significantly increased in some organs in the vaccinated trout. The results indicate that the survival of the vaccinated fish after bacterial challenge seems to be correlated with an ability to clear bacterial infection over time. Additionally, the results indicate that immersion vaccines based on Y. ruckeri serotype O1, biotype 2 confers significant cross protection against biotype 1.