Cystoisospora suis causes neonatal diarrhea in piglets worldwide. Although histopathological identification of this parasite is difficult owing to its similarity with other enteric coccidian parasites, including Eimeria spp., information regarding its molecular epidemiology in Japan is lacking. In this study, a 66-day-old post-weaned piglet reared in Shizuoka Prefecture, Japan, showed diarrhea and was examined using parasitological, histopathological, ultrastructural, and molecular methods to characterize the pathogens of the disease. Gross lesions were characterized by emaciation, thinning of the small intestinal wall, and lack of pulmonary collapse. Histopathologically, severe villous atrophy was detected in the jejunum, and Cystoisospora-like parasites were found in the cytoplasm of the epithelium. Interstitial pneumonia, purulent meningitis, pericarditis, and lymphatic abscesses were also noted. A microbiological and immunohistochemical analysis revealed that the piglet was also infected with porcine circovirus type 2, Escherichia coli serogroup O8, and group A rotavirus. Coccidial oocysts were recovered from diarrheic feces. PCR analysis targeting the rRNA internal transcribed spacer 1 (ITS1) and cytochrome c oxidase subunit 1 (COX1) genes of C. suis and sequencing revealed 100% similarity to those of C. suis in Kumamoto Prefecture, Japan. This study is the first report on the molecular identification of C. suis COX1 genes in Japan.