Piwi-interacting RNAs (piRNAs), a kind of small non-coding RNAs (ncRNAs), play pivotal parts in maintaining the genomic stability and modulating biological processes such as growth and development via regulation of gene expression. However, piRNAs in Asian honey bee (Apis cerana) is still largely unknown at present. In this current work, on basis of previously gained high-quality small RNA-seq datasets, piRNAs in the larval guts of Apis cerana cerana, the nominate species of A. cerana, was for the first time identified, followed by in-depth investigation of the regulatory roles of differentially expressed piRNAs (DEpiRNAs) in the developmental process of the A. c. cerana. Here, a total of 621 piRNAs were identified in the A. c. cerana larval guts, among which 499 piRNAs were shared by 4- (Ac4 group), 5- (Ac5 group), and 6-day-old (Ac6 group) larval guts, while the numbers of unique ones were 79, 37, and 11, respectively. piRNAs each group were ranged from 24 nt to 33 nt in length, and the first base of piRNAs had a cytosine (C) bias. Additionally, five up-regulated and five down-regulated piRNAs were identified in the Ac4 vs. Ac5 comparison group, 9 of which could target 9, 011 mRNAs; these targets were involved in 41 GO terms and 137 pathways. Comparatively, 22 up-regulated piRNAs were detected in the Ac5 vs. Ac6 comparison group, 21 of which could target 28, 969 mRNAs; these targets were engaged in 46 functional terms and 164 pathways. The results suggested the overall alteration of expression pattern of piRNAs during the developmental process of A. c. cerana larvae. Regulatory network analysis showed that piR-bmo-748815 and piR-bmo-512574 in the Ac4 vs. Ac5 comparison group as well as piR-bmo-716466 and piR-bmo-828146 in the Ac5 vs. Ac6 comparison group linked to the highest number of targets. Further investigation indicated that targets of DEpiRNAs in the above-mentioned two comparison groups could be annotated to several growth and development-associated pathways, such as Jak/STAT, TGF-β, and Wnt signaling pathways, indicating the involvement of DEpiRNAs in modulating larval gut development via these crucial pathways. Moreover, the expression trends of six randomly selected DEpiRNAs were verified using a combination of stem-loop RT-PCR and RT-qPCR. These results not only provide a novel insight into the development of the A. c. cerana larval guts, but also lay a foundation for uncovering the epigenetic mechanism underlying the larval gut development.