MicroRNAs (miRNAs) are a class of small noncoding RNAs that have major functions in the development and progression of colorectal cancer (CRC) as tumor suppressors or oncogenes. The aim of the current research was to assess the role of miR-485-3p and miR-4728-5p in the pathogenesis of CRC. In this study, fresh tumor and adjacent non-tumor tissue samples were obtained from a total of 59 CRC patients, 37 from colon and 22 from rectum. The expression profiles of miR-485-3p and miR-4728-5p were determined using qRT-PCR. miRNA-related transcription factor (TF) regulatory networks were constructed using the TransmiR v2.0, TF-regulated target genes were determined using the Human.mirFFL.DB and TRRUST v2.0, functional annotation and pathway enrichment analyses were performed using DIANA-mir-Path v3.0 and -Tarbase v7.0. The results demonstrated that the expression levels of both miR-485-3p and miR-4728-5p were very significantly downregulated in CRC tissues (fold changes = 0.42 ± 0.70 and 0.59 ± 1.06, respectively; both p = 0.000). On the other hand, lower expression levels of miR-485-3p were detected in the both rectum and colon. Moreover, the decrease in the expression levels of miR-4728-5p was correlated with increasing age. However, these differences were not statistically significant according to the FDR-related p-values (0.126 and 0.168, respectively). By bioinformatics analyses, miR-485-3p and miR-4728-5p-related TFs were identified. Some of these TFs, namely, AR, CREB1, CEBPB, FOXA1, GTF2I, MAZ, NCOR2, NFIC, NRF1, SIN3A, SREBF1, SREBF2, p53 and YY1, appeared to be associated with CRC and were, therefore, selected to construct miRNA-TF-gene networks of potential targets for the early diagnosis and treatment of CRC. Pathway enrichment analysis indicated Hippo signaling pathway as heavily regulated by miR-485-3p. It seems that the decrease in expression levels of miR-485-3p and miR-4728-5p might be associated with development of colorectal cancer.