Objective To investigate the expression and clinical significance of P-element-induced wimpy testis (Piwi)-interacting RNAs (piRNAs) in childhood nephroblastoma. Methods The differential piRNAs of Piwil2-iCSC (P-element-induced wimpy testis like 2[Piwil2] reprogramming human fibroblast) were screened by high-throughput sequencing. Target genes were predicted by miRanda software and performed by gene ontology (GO) function analysis. Differential piRNAs and their target genes were detected by qRT-PCR in tumor tissues and normal kidney tissues of 34 children with nephroblastoma. The correlation of differential piRNAs and their target genes with clinicopathological characteristics of nephroblastoma was analyzed. Results A total of 230 differential piRNAs were screened out through high-throughput sequencing, and 43 target genes were predicted by miRanda. The GO analysis showed that the biological process of target genes was mainly involved in regulating the cytosolic calcium ion concentration and its molecular function was mainly involved in the activity of ATPase and poly (A) RNA binding. The expression of five unknown differentially expressed piRNAs and their target genes were detected in nephroblastoma and normal kidney tissues. The expression of NU13 (13th unknown upregulated piRNA) and NU9 (9th unknown upregulated piRNA) were significantly downregulated in tumor tissues (both P<0.01), while the expression of their target genes NOP56 ribonucleoprotein (NOP56, P=0.58) and 40S ribosomal protein S8 (RPS8, P=0.29) had no significant difference between tumor tissues and normal kidney tissues. The expression of ND5, ND7, ND9 (5th, 7th, 9th unknown downregulated piRNA) and their target gene MT-RNR2 like protein 1 (MTRNR2L1) were significantly downregulated in tumor tissues (all P<0.01). The above-mentioned piRNAs and their target genes had no significant correlation with age, gender, tumor stage, or pathological type of the children (all P>0.05). Conclusion piRNA NU9, NU13, ND5, ND7, ND9 and the target gene MTRNR2L1 are differentially expressed in children with nephroblastoma. They are expected to be markers to distinguish nephroblastoma from normal kidney tissues.