We have previously established that mucosal uptake of recombinant fowlpox virus (rFPV) vaccines is far superior to other vector-based vaccines. Specifically， intranasal priming with rFPV vaccines can recruit unique antigen-presenting cells， which induce excellent mucosal and systemic HIV-specific CD8+ T-cell immunity. In this study， we have for the first time investigated the in vivo dissemination， safety and expression kinetics of rFPV post intranasal delivery using recombinant viruses expressing green fluorescent protein or mCherry. Both confocal microscopy of tissue sections using green fluorescent protein and in vivo Imaging System (IVIS) spectrum live animal and whole organ imaging studies using mCherry revealed that (i) the peak antigen expression occurs 12 to 24 h post vaccination and no active viral gene expression is detected 96 h post vaccination. (ii) The virus only infects the initial vaccination site (lung and nasal cavity) and does not disseminate to distal sites such as the spleen or gut. (iii) More importantly， rFPV does not cross the olfactory receptor neuron pathway. Collectively， our findings indicate that rFPV vector-based vaccines have all the hallmarks of a safe and effective mucosal delivery vector， suitable for clinical evaluation.