© 2015 Segura et al. Background: Cryptosporidium infection is a worldwide cause of diarrheal disease. To gain insight into the epidemiology of the infection in a certain geographic area, molecular methods are needed to determine the species/genotypes and subtypes. Methodology/Principal Findings: From 2004 to 2009, 161 cryptosporidiosis cases were detected in two hospitals in Barcelona. Diagnosis was performed by microscopic observation of oocysts in stool specimens following modified Ziehl-Neelsen staining. Most cases (82%) occurred in children. The number of cases increased in summer and autumn. Molecular characterization of Cryptosporidium was performed in 69 specimens, and C. hominis and C. parvum were identified in 88.4% and 10.1%of the cases, respectively. C. meleagridis was detected in one specimen. Subtyping based on the gp60 polymorphism showed six subtypes, four C. hominis and two C. parvum. Subtype IbA10G2 was the most prevalent subtype corresponding to 90% of all C. hominis isolates. This is the first report on the distribution of specific Cryptosporidium subtypes from humans in Spain. Conclusions/Significance: In our geographic area, the anthroponotic behavior of C. hominis, the lower infective dose, and the higher virulence of certain subtypes may contribute to the high incidence of human cryptosporidiosis caused by the IbA10G2 subtype. Further studies should include populations with asymptomatic shedding of the parasite.