Animals have evolved various sex determination systems. Here, we describe a newly found mechanism. A long noncoding RNA (lncRNA) transduces complementary sex determination (CSD) signal in the invasive Argentine ant. In this haplodiploid species, we identified a 5-kilobase hyper-polymorphic region underlying CSD: Heterozygous embryos become females, while homozygous and hemizygous embryos become males. Heterozygosity at the
CSD
locus correlates with higher expression of
ANTSR
, a gene that overlaps with the
CSD
locus and specifies an lncRNA transcript.
ANTSR
knockdown in
CSD
heterozygotes leads to male development. Comparative analyses indicated that, in Hymenoptera,
ANTSR
is an ancient yet rapidly evolving gene. This study reveals an lncRNA involved in genetic sex determination, alongside a previously unknown regulatory mechanism underlying sex determination based on complementarity among noncoding alleles.