Xenotransplantation from pigs to humans holds great promise for addressing organ shortages. However, immunological barriers, including hyperacute rejection mediated by α-Gal epitopes and macrophage phagocytosis, must be overcome. To this end, we generated genetically modified pigs with targeted knock-in of human heme oxygenase-1 (HO1) and CD47 expression cassettes into the exon 4 of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in a GGTA1-knockout (GTKO) background. Initial analysis of GT, CMAH, and B4GAL expression across various porcine tissues revealed unexpectedly low endogenous CMAH expression levels, prompting us to utilize alternative promoters for transgene expression. We employed the human HO1 promoter to drive regulated HO1 expression, considering its potent biological activity and potential for adverse effects upon overexpression. Constitutive, high-level expression of human CD47, a "don't eat me" signal, was achieved using the previously established pEF1α promoter. A bicistronic vector encoding HO1 and CD47 was constructed and demonstrated inducible expression upon PMA and human serum treatment in porcine cells, confirming its regulatory capacity. The knock-in vector was precisely integrated into the CMAH locus of GTKO porcine cells using CRISPR/Cas9 technology. Somatic cell nuclear transfer resulted in the generation of two cloned piglets, with one surviving for one week. Post-mortem analysis of the deceased piglet's heart, kidney, liver, lung, and spleen by Western blotting confirmed the presence of both human HO1 and CD47 proteins in all examined tissues. Immunohistochemistry (IHC) analysis showed HO1 expression predominantly in the liver and lung. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) from the surviving piglet demonstrated the expression of human CD47 on the cell surface. These results demonstrate the successful targeted knock-in of human HO1 and CD47 into the CMAH locus of GTKO pigs, achieving transgene expression in vital organs. The use of specific promoters allows for regulated HO1 expression and high-level CD47 expression, representing a significant step towards generating donor pigs with enhanced resistance to immune rejection for xenotransplantation. Further studies on the surviving pig and subsequent generations will be crucial to evaluate the long-term efficacy and safety of this genetic modification strategy.