Supergenes control alternative social organization in at least six independent ant lineages, raising novel questions about the genetic basis of the behavioural processes that lead to polygynous (multiple-queen) colonies. Here, we investigated whether a social supergene influences worker behaviour, queen behaviour and colony growth in the Alpine silver ant, Formica selysi . This species exhibits both monogynous (single-queen) and polygynous colonies, with a large, inverted supergene haplotype ( P ) exclusively present in individuals from polygynous colonies. We crossed queens originating from monogynous colonies ( MM supergene genotype) with males originating either from monogynous colonies ( M haplotype) or polygynous colonies ( P haplotype). We then allowed queens to establish incipient, single-queen colonies in the laboratory. After 1 year, single MM queens mated to P males had more worker offspring than single MM queens mated to M males, consistent with a heterozygote advantage. We then tested whether the workers would accept non-nestmate queens introduced into their colony. Contrary to our expectations, we found no evidence that the P haplotype made workers more likely to accept additional queens. Host workers consistently attacked and rejected foreign queens, irrespective of worker supergene genotype. Furthermore, MM and MP workers had similar cuticular lipid profiles. However, in contrast to workers, queens of polygynous origin (carrying the P haplotype) behaved differently from queens of monogynous origin. When introduced into a foreign host colony, queens of polygynous origin were more agitated, performed fewer appeasement behaviours and received more acts of aggression from workers than queens of monogynous origin. In colony-founding assays, queens originating from polygynous colonies were more likely to peacefully associate in the same nest. These findings suggest that in a socially polymorphic Formica species, the behaviour of queens plays a more important role than previously recognized in determining colony social structure, while the role of workers is context dependent. • We tested how a supergene controlling colony queen number impacts ant behaviour. • Workers rejected non-nestmate queens, irrespective of worker supergene genotype. • Queens from polygynous colonies associated more during colony founding. • Our findings indicate that queen behaviour influences colony social structure. • The impact of the social supergene is context dependent.