Re-vaccination against canine adenovirus (CAV) is performed in ≤3-year-intervals but their necessity is unknown. The study determined anti-CAV antibodies within 28 days of re-vaccination and factors associated with the absence of antibodies and vaccination response. Ninety-seven healthy adult dogs (last vaccination ≥12 months) were re-vaccinated with a modified live CAV-2 vaccine. Anti-CAV antibodies were measured before vaccination (day 0), and after re-vaccination (day 7, 28) by virus neutralization. A ≥4-fold titer increase was defined as vaccination response. Fisher's exact test and multivariate regression analysis were performed to determine factors associated with the absence of antibodies and vaccination response. Totally, 87% of dogs (90/97; 95% CI: 85.61-96.70) had anti-CAV antibodies (≥10) before re-vaccination. Vaccination response was observed in 6% of dogs (6/97; 95% CI: 2.60-13.11). Time since last vaccination (>3-5 years, OR = 9.375, p = 0.020; >5 years, OR= 25.000, p = 0.006) was associated with a lack of antibodies. Dogs from urban areas were more likely to respond to vaccination (p = 0.037). Many dogs had anti-CAV pre-vaccination antibodies, even those with an incomplete vaccination series. Most dogs did not respond to re-vaccination. Based on this study, dogs should be re-vaccinated every 3 years or antibodies should be determined.