Background: Re-vaccination against canine adenovirus (CAV) is performed in ≤3-year-intervals but its 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. Methods: 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. Results: 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). Conclusion: 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.