The purposes of this study were to investigate the role of Aplysia Ras Homolog I (ARHI) on cell growth, proliferation, apoptosis, and other biological characteristics of HER2-positive breast cancer cells. Our goal was to provide experimental evidence for the development of future effective treatments of HER2-positive breast cancer. A pcDNA3.1-ARHI eukaryotic expression vector was constructed and transfected into the human HER2-positive breast cancer cell lines SK-BR-3 and JIMT-1. Then, various experimental methods were utilized to analyze the biological characteristics of ARHI-expressing breast cancer cells and to examine the impact of expression of the ARHI gene on cyclin D1, p27(Kip1), and calpain1 expression. We further analyzed the cells in each group after treatment with trastuzumab to examine the effects of this drug on various cellular characteristics. When we compared pcDNA3.1-ARHI-expressing SK-BR-3 and JIMT-1 cells to their respective empty vector and control groups, we found that cell viability was significantly lower (p < 0.05) in the ARHI-expressing cells, and the proportions of G1 phase cells and apoptotic cells were significantly higher in the ARHI-expressing cells (p < 0.05). In all groups of SK-BR-3 cells, trastuzumab treatment significantly decreased cell growth (p < 0.05). The proportion of cells in G1 phase and the number of apoptotic cells in the pcDNA3.1-ARHI-expressing group were significantly higher than that in the empty vector group and the control group (p < 0.05). The growth of pcDNA3.1-ARHI-transfected JIMT-1 cells was significantly decreased (p < 0.05), while the proportion of apoptotic cells was significantly increased (p < 0.05). Cell growth, viability, and the percentage of apoptotic cells were similar between the JIMT-1 empty vector and control groups. ARHI expression inhibited cyclin D1 expression in SK-BR-3 cells and JIMT-1 cells, while it promoted p27(Kip1) and calpain1 expression in these cells. ARHI expression inhibits the growth and proliferation of HER2-positive breast cancer cells, while it also promotes apoptosis in these cells. ARHI expression also improves the sensitivity of JIMT-1 cells to trastuzumab by inducing apoptosis.