Immune function is likely to be shaped by multiple infections over time. Infection with one pathogen can confer cross-protection against heterologous pathogens. We tested the hypothesis that latent murine gammaherpesvirus 68 (gammaHV68) infection modulates host inflammatory responses and susceptibility to mouse adenovirus type 1 (MAV-1). Mice were infected intranasally (i.n.) with gammaHV68. 21 days later, they were infected i.n. with MAV-1. We assessed cytokine and chemokine expression by quantitative reverse transcriptase real-time PCR, cellular inflammation by histology, and viral loads by quantitative real-time PCR. Previous gammaHV68 infection led to persistently upregulated IFN-gamma in lungs and spleen and persistently upregulated CCL2 and CCL5 in the lungs. Previous gammaHV68 infection amplified MAV-1-induced CCL5 upregulation and cellular inflammation in the lungs. Previous gammaHV68 infection was associated with lower MAV-1 viral loads in the spleen but not the lung. There was no significant effect of previous gammaHV68 on IFN-gamma expression or MAV-1 viral loads when the interval between infections was increased to 44 days. In summary, previous gammaHV68 infection modulated lung inflammatory responses and decreased susceptibility to a heterologous virus in an organ- and time-dependent manner.