The induction efficiency of maize embryonic callus is highly dependent on the genotype, and only a few lines possess a high capacity for callus formation. Although certain genes and pathways have been reported to contribute to the regulation of callus induction, to the best of our knowledge, the functions of the small interfering RNAs (siRNAs) involved in this process remain unknown. In this study, we identified 861 differentially expressed siRNAs and 576 target genes in the callus induction process. These target genes were classified into 3 clusters, and their functions involve controlling metalloexopeptidase activity, catalase activity, transcription regulation, and O-methyltransferase activity. In addition, certain genes related to auxin transport and stem cell or meristem development (e.g., PLT5-like, ARF15, SAUR-like, FAS1-like, Fea3, SCL5, and Zmwox2A) were regulated by the differentially expressed siRNAs. Moreover, zma-siR004119-2 directly cleaves the 5' UTR of Homeobox-transcription factor 25, which further leads to the down-regulation of its expression. Twelve 24-nt-siRNAs led to the hyper-methylation of GRMZM2G013465, which further decreases its expression. These results suggest that differentially expressed siRNAs regulate callus formation by controlling the expression of their target genes.