We identified a probable new null HLA‐C allele, C*03:23N, which originated from C*03:04:01:02, but does not react with Cw3 antibodies. This allele was identified by sequence analysis, which indicated that a single G‐to‐A substitution at position 406 in exon 3 created a null allele under a new mechanism: the mutation changes the position of the intron 2‐exon 3 splice site to be further into exon 3, leading to a frameshift and a premature stop codon. Sequence analysis of cDNA confirmed the existence of the causative alternative acceptor splice site and the resultant deletion of 64 nucleotides in exon 3. Analysis of 220 blood or bone marrow donors in Japan with C*03:23N demonstrated that Japanese HLA‐C*03:23N is on the haplotype A*26:01∼C*03:23N∼B*40:02∼DRB1*09:01.