MicroRNAs (miRs) are small, non-coding RNAs that negatively regulate gene expression. The stem-loop sequence miR-101-1 generates mature miR-101-5p and miR-101-3p. The function and target mRNA of miR-101-5p have not yet been elucidated in detail. Here, we demonstrate that miR-101-5p inhibits the expression of RAP1A, a member of the RAS gene family. Transfection of a miR-101-5p mimic significantly inhibited the expression of RAP1A mRNA in HeLa, HEK293, A549, and COLO201 cells. The same treatment significantly inhibited cell proliferation. The cytostatic effect with transfection of miR-101-5p was antagonized by treatment with the RAP inhibitor salirasib. These results suggested that miR-101-5p inhibits RAP1A, and thus, the expression levels of miR-101-5p regulate cell proliferation.