Objective The incidence of infective endocarditis (IE) is increasing, as is the insertion of prosthetic heart valves. We aimed to examine nationwide temporal trends in the incidence of IE in patients with a prosthetic heart valve in Denmark from 1999 to 2018. Methods Using the Danish nationwide registries, we identified patients who underwent heart valve implantation (for other reasons than IE) between 1999 and 2018. Crude incidence rates of IE per 1000 person-years (PY) were computed in 2-year intervals. IE incidences were compared using sex-adjusted and age-adjusted incidence rate ratios (IRR) using Poisson regression across calendar periods (1999-2003, 2004-2008, 2009-2013, and 2014-2018). Results We identified 26 604 patients with first-time prosthetic valve implantation (median age 71.7 years (IQR 62.7-78.0), 63% males). The median follow-up time was 5.4 years (IQR 2.4-9.6). Patients in the time period 2014-2018 were older (median age of 73.9 years (66.2:80.3)), and with a higher burden of comorbidities compared with the time period 1999-2003 (median age of 67.9 years (58.3:74.5)) at the time of implantation. A total of 1442 (5.4%) patients developed IE. The lowest IE incidence rate was 5.4/1000 PY (95% CI 3.9 to 7.4) in 2001-2002, and the highest incidence rate was 10.0/1000 PY (95% CI 8.8 to 11.1) in 2017-2018 with an unadjusted increase during the study period (p=0.003). We found an adjusted IRR of 1.04 (95% CI 1.02 to 1.06) (p<0.0007) per two calendar-years increments. Age-adjusted IRR for men were 1.04 (95% CI 1.01 to 1.07) (p=0.002) per two calendar years increment, and for women 1.03 (95% CI 0.99 to 1.07) (p=0.12), with p=0.32 for interaction. Conclusion In Denmark, the incidence of IE increased during the last 20 years in patients with prosthetic heart valves.