Porcine epidemic diarrhea virus (PEDV) is a causative agent of porcine intestinal disease, which causes vomiting, diarrhea, and dehydration in piglets. PEDV is associated with the most severe pathogenesis in one-week-old piglets, with mortality rates reaching 100%. A PEDV strain was isolated from the intestinal tract of diarrheic piglets from a pig farm in Jiangsu Province in March 2016, termed the JS201603 isolate. The isolated virus was confirmed to be PEDV via RT-PCR, electron microscopy, a cytopathic effect assay and sequence analysis. The S and ORF3 genes of the JS201603 isolate were sequenced, revealing that the S gene was associated with a 15-base insertion at 167 nt, 176 - 186 nt, and 427 - 429 nt, as well as a six-base deletion in 487 - 492 nt, indicating that it was a current epidemic variant compared with the classical strain, CV777. No deletion occurred between 245 - 293 nt of the ORF3 gene in the JS201603 isolate compared with the vaccine isolates YY2013 and SQ2014. An experimental infection model indicated that the piglets in the challenge group successively developed diarrhea, exhibiting yellow-colored loose stools with a foul odor. The piglets in the JS201603 isolate challenge group displayed reduced food consumption, lost weight, and in severe cases even died. No abnormalities were observed in the control group. The JS201603 variant isolated in this study contributes to the evolutionary analysis of diarrhea virus. The experimental infection model has established a foundation for further studies on vaccine development.