Mating causes dramatic changes in female insects at the behavioural, physiological and molecular level. The factors driving these changes (e.g. seminal proteins, seminal volume) and the molecular pathways by which these factors are operating have been characterized only in a handful of insect species. In the present study, we use instrumental insemination of honey bee queens to examine the role of the insemination substance and volume in triggering post-mating changes. We also examine differences in gene expression patterns in the fat bodies of queens with highly activated ovaries to determine if events during copulation can cause long-term changes in gene expression. We found that the instrumental insemination procedure alone caused cessation of mating flights and triggered ovary activation, with high-volume inseminated queens having the greatest ovary activation. Hierarchical clustering grouped queens primarily by insemination substance and then insemination volume, suggesting that while volume may trigger short-term physiological changes (i.e. ovary activation) substance plays a greater role in regulating long-term transcriptional changes. The results of gene ontology analysis and comparison with previous studies suggest that both insemination substance and volume trigger molecular post-mating changes by altering overlapping gene pathways involved in honey bee reproduction. We also discuss the effects on two genes (vitellogenin and transferrin) involved in reproduction and defence responses.