Abstract We investigated seroprevalence and factors associated with Leptospira spp. infections in humans in rural Northern Germany. Sera of 450 participants were tested for leptospira-reactive IgG antibodies by two enzyme-linked immunosorbent assays (ELISA). A narrow (specific) and a broad (sensitive) case definition were applied and results compared in the analysis. Personal data were collected via questionnaire and associations with the serostatus were investigated by multivariable logistic regression. The seroprevalence estimates were 1.6% (95%-confidence interval (CI) = 0.63–3.2) under the narrow and 4.2% (95%-CI = 2.6–6.5%) under the broad case definition. Few (14%) participants knew about the pathogen. No seropositive participant recalled a prior leptospirosis diagnosis. Spending more than two hours a week in the forest was significantly associated with anti-leptospira IgG in both models (broad case definition: adjusted odds ratio (aOR) = 2.8, 95%-CI = 1.2–9.1; narrow case definition: aOR = 11.1, 95%-CI = 1.3–97.1). Regular cleaning of storage rooms was negatively associated in the broad (aOR = 0.17, 95%-CI = 0.03–0.98) and touching a dead rodent in the past 10 years in the narrow case definition model (aOR = 0.23, 95%-CI = 0.05–1.04). Our findings support risk factors identified in previous investigations. To counter the low awareness for the pathogen, we recommend that health authorities communicate risks and preventive measures to the public by using target-group specific channels.