Transgenic cotton expressing insecticidal proteins from Bacillus thuringiensis (Bt) has been grown in Australia for over 20 years and resistance remains the biggest threat. The native moth, Helicoverpa punctigera is a significant pest of cotton. A genotype causing resistance to Cry1Ac in H. punctigera was isolated from the field and a homozygous line established. The phenotype is recessive and homozygous individuals possess 113 fold resistance to Cry1Ac. Individuals that carry Cry1Ac resistance genes are rare in Australia with a frequency of 0.033 being detected in field populations. RNAseq, RT-PCR and DNA sequencing reveals a single nucleotide polymorphism at a splice site in the cadherin gene as the causal mutation, resulting in the partial transcription of the intron and a premature stop codon. Analysis of Cry1Ac binding to H. punctigera brush border membrane vesicles showed that it is unaffected by the disrupted cadherin gene. This suggests that the major Cry1Ac target is not cadherin but that this molecule plays a key role in resistance and therefore the mode of action. This work adds to our knowledge of resistance mechanisms in H. punctigera and the growing literature around the role of cadherin in the mode of action of Cry1 type Bt proteins.