Humans are infected by Toxoplasma gondii worldwide and its consequences may seriously affect an immune deprived population such as HIV and transplanted patients or pregnant women and foetuses. A deep knowledge of toxoplasmosis seroprevalence in Spain is needed in order to better shape health policies and educational programs. We present the results of the first systematic review and meta-analysis on the human prevalence for this disease in Spain. Databases (PubMed, Web of Science, SCOPUS and Teseo) were searched for relevant studies that were published between January 1993 and December 2023 and all population-based cross-sectional and longitudinal studies reporting the human seroprevalence in Spain were revised. Within the population analysed, our targeted groups were immunocompetent population, pregnant women and immunocompromised patients. Among 572 studies and 35 doctoral theses retrieved, 15 studies and three doctoral theses were included in the meta-analysis. A random effects model was used for the meta-analyses due to the high heterogeneity found between studies (I2: 99.97), since it is a statistically conservative model, in addition to allowing better external validity. The global pooled seroprevalence was 32.3% (95% CI 28.7–36.2%). Most of the studies carried out were in pregnant women and the meta-analysis reported that the pooled seroprevalence of toxoplasmosis in pregnant women in Spain was 24.4% (24,737/85,703, 95% CI 21.2–28.0%), based on the random effects model. It is recommended to continue monitoring the seroprevalence status of T. gondii in order to obtain essential guidelines for the prevention and control of the infection in the population.