Purpose: miR-100-5p has been found upregulated in Oxaliplatin-resistant LoVo colorectal cell lines, but its function still need further study. Here, we investigated its function in cell regulation chemoresistance in chemoresistant colorectal cancer cell lines.Methods: Microarray, quantitative polymerase chain reaction (qRT-PCR) and westernblot were used to analysis the level of miR-100-5p. The downstream candidate gen target of miR-100-5p, CTDSPL in Oxaliplatin resistant (OXR)-LoVo colorectal cell lines were screened using the different types of bioinformatics based miRNA target prediction data are TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/ and mirTar (http://mirtar.mbc.nctu.edu.tw/human/). CTDSPL, a phosphatase-like tumor suppressor level was detected by westernblot as well with cell cycle progression chemoresistance associated colorectal cancer cell CTDSPL/p-RB/E2F1. Immunofluorescence assay and westernblot showed the cell properties were altered between parental and Oxaliplatin resistant LoVo cell lines. Bioinformatic prediction, (http://alggen.1si.upc.es/cgi-bin/promo_v3) was used to extend the investigation of miR-100-5p upstream regulation through checking dissimilarity index with margin less or equal than 15%, FOXP3, trancription factor forkhead box as upstream target. Interaction between miR-100-5p and CTDSPL regulation was confirmed using mimic miR-100-5p overexpression and knockdown upstream target sh-RNA approach.Results: hsa-miR-100-5p level was found significantly upregulated in LoVo-OXR than parental LoVo cell line, resulted in downregulation CTDSPL as a major target of miR-100-5p. The downregulated of CTDSPL/p-RB/E2F1 promotes cell proliferation and cell cycle progression in LoVo OXR compared with LoVo parental cell lines. Moreover, among various upstream targets FOXP3 is an upstream regulator of miR-100-5p. In addition, mimic miR-100-5p overexpression downregulated CTDSPL/p-RB/E2F1 signaling pathway was reverted upon miR-100-5p inhibition in CRC cell lines.Conclusion: Collectively, miR-100-5p is chemoresistance inducer through modulation of CTDSPL/p-RB/E2F1 signaling pathway in CRC cell. This phenomenon might offer a therapeutic platform for overcoming chemoresistance in cancer treatment.