Cyclin-dependent kinase-like 1 (CDKL1) is a member of cell division control protein 2 (CDC2)-related serine-threonine protein kinase family, and it is likely to occur in malignant tumors, plays an important impact on the progress. This study aimed to evaluate the expression of CDKL1 in breast cancer and regulation in cancer cell growth. In the work, the CDKL1 mRNA level in fresh biopsy tissues from 186 breast cancer patients, with 98 benign tissues as negative control, and CDKL1 protein in 30 paraffin-embedded tissues from primary breast cancer patients were detected by the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay and immunohistochemical staining, respectively. The roles of CDKL1 in cell growth were analyzed with CDKL1 short hairpin RNA (shRNA) inhibitor-transfected cells. CDKL1 was overexpressed in breast cancer patients and had a positive detection efficiency of 77% (144/186), which showed statistically significant difference compared with estrogen receptor (ER), progesterone receptor (PR), P53, vascular endothelial growth factor (VEGF), and E-cadherin (E-cad) (p<0.05). Inhibiting CDKL1 function with shRNA, MCF-7 cells exhibited obvious accumulation at the G2/M phase and increased sensitivity to cell cycle chemotherapeutic drugs. The results suggested that the CDKL1 gene could be a potential tumor marker for diagnosis and a gene target for therapy.