Protein　kinase　CK2　is　considered　as　an　emerging　target　in　cancer　therapy,　and　recent　efforts　have　been　made　to　develop　its　ATP-competitive　inhibitors,　but　achieving　selectivity　with　respect　to　related　kinases　remains　challenging　because　of　the　highly　conserved　ATP-binding　pocket　of　kinases.　Non-ATP　competitive　inhibitors　might　solve　this　challenge;　one　such　strategy　is　to　identify　compounds　that　target　the　CK2　alpha/CK2　beta　interface　as　CK2　holoenzyme　antagonists.　Here　we　improved　the　binding　affinity　to　CK2　alpha　and　cell-based　anti-cancer　activity　of　a　CK2　beta-derived　cyclic　peptide　(Pc)　by　combining　structure-based　computational　design　with　experimental　evaluation.　By　analyzing　molecular　dynamics　simulations　of　Pc　bound　to　CK2　alpha,　a　series　of　Pc-derived　peptides　was　rationally　designed　and　synthesized　to　evaluate　their　binding　affinity　to　CK2　alpha,　as　well　as　anti-proliferative　and　pro-apoptotic　effects　against　HepG2　cancer　cell　line.　One　amino　acid　substitutions　on　Pc,　I192F,　exhibited　over　10-fold　improvement　in　the　predicted　binding　affinity　to　CK2　alpha　when　compared　to　Pc,　and　a　cell-permeable　version,　I192F-Tat,　also　demonstrated　more　potent　anti-proliferative　and　pro-apoptotic　effects　against　HepG2　compared　to　Pc.　A　second　modification　of　Pc,　H193W,　also　led　to　more　potent　cell-based　activity,　despite　having　weaker　binding　affinity　(similar　to　5x)　to　CK2　alpha.　The　discovery　of　the　I192F　and　H193W　peptides　provides　new　insights　for　further　optimization　of　CK2　antagonist　candidates　as　anti-cancer　leads.