Background: Human rabies, often contracted through dog bites, is a serious but neglected public health problem in the tropics, including Ghana. Due to its high fatality rate, adequate knowledge and vaccination of domestic dogs against the disease are very crucial in reducing its burden. We examined dog owners’ knowledge level on rabies and factors that influenced anti-rabies vaccination of dogs in the Upper East Region of Ghana. Methods: This descriptive cross-sectional study was conducted among 260 randomly sampled dog owners in six communities from six Districts using a multistage sampling technique, in the Upper East Region of Ghana. An interviewer-administered questionnaire was used to collect data from the respondents. Descriptive and inferential analyses were done using STATA 14.1. Results: While knowledge about rabies was 199 (76.5%), that about anti-rabies vaccination was 137 (52.7%). District of residence (χ2 = 112.59, p < 0.001), sex (χ2 = 6.14, p = 0.013), education (χ2 = 20.45, p < 0.001) as well as occupation (χ2 = 11.97, p = 0.007) were significantly associated with rabies knowledge. District of residence (χ2 = 57.61, p < 0.001), Educational level (χ2 = 15.37, p = 0.004), occupation (χ2 = 11.66, p = 0.009), religion (χ2 = 8.25, p = 0.016) and knowledge on rabies (χ2 = 42.13, p < 0.001) were also statistically associated with dog vaccination against rabies. Dog owners with good knowledge on rabies for instance, were more likely to vaccinate their dogs against rabies compared to those with poor knowledge [AOR = 1.99 (95% CI: 0.68, 5.86), p = 0.210]. Dog owners with tertiary level of education were also 76.31 times more likely (95% CI: 6.20, 938.49, p = 0.001) to have good knowledge about rabies compared to those with no formal education. Conclusions: Dog owners in the Upper East Region of Ghana had good knowledge about rabies. This, however, did not translate into correspondingly high levels of dog vaccination against the disease. Rabies awareness and vaccination campaigns should, therefore, be intensified in the region, especially among the least educated and female dog owners.