Objective To describe the phenotype of NK cells in Bama miniature pigs, and establish an efficient activation and culture method for porcine cytokine-induced killer (CIK) cells in vitro. Methods The porcine peripheral blood mononuclear cells (PBMCs) were isolated by Percoll gradient centrifugation, and the phenotype of NK cells was tested by detecting the CD2⁺/CD8⁺/CD3^- cell compartment. To establish an efficient activation and culture method for porcine CIK cells, we optimized the culture conditions to improve the CIK activation efficiency. Results Using the optimized induction culture conditions, the ratio of CIK (CD2⁺/CD8⁺/CD3^-) cells was up to 43.63% at the fifth day, approximately 5.59 times increased compared with the initially separated PBMCs. Cell proliferation experiments showed that three obvious fluorescence peaks were observed on the fifth day. The results indicated that the induced CIK cells underwent three times cell division, in theory, about increased 8-fold compared with the initial separation of PBMCs. Furthermore, the qRT-PCR result of the surface markers of porcine NK cells also showed a similar variation tendency as the flow cytometry results. Conclusions Our findings demonstrate the successful establishment of an efficient activation and culture method for porcine CIK cells in vitro.