The Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), is the most destructive insect pest of potato, Solanum tuberosum (L.), in North America. Avidin sequesters available biotin, thereby causing abnormal growth and development of insects. We expressed avidin in two potato lines: MSE149-5Y, a susceptible potato line, and ND5873-15, a line with S. chacoense-derived insect resistance. A preliminary study was conducted to determine the bioactivity of the transgene in each background. A single transgenic line was selected in each background for further studies. Detached leaf bioassays were performed on transgenic and nontransgenic clones of the susceptible and S. chacoense lines by using first-stage Colorado potato beetle larvae. Consumption, survival, and survivor growth were measured after 5 d. Larvae consumed significantly less on the two avidin-expressing lines compared with the nontransgenic lines. Survival was also significantly less for larvae feeding on transgenic avidin lines compared with the nontransgenic lines. The mass of survivors was significantly reduced on two transgenic avidin lines compared with the nontransgenic lines. Further studies examined the development from first-stage larvae to adulthood on greenhouse-grown whole plants in a no-choice setting for larvae fed on the four potato lines. Development from first stage to pupation was significantly prolonged for larvae fed on the avidin line compared with larvae fed on the susceptible line. Significantly fewer larvae fed on transgenic avidin plants, avidin or avidin S. chacoense-derived line survived to adulthood compared with survival on nontransgenic plants, susceptible or S. chacoense-derived line. Avidin-based resistance may be useful in managing Colorado potato beetle populations in commercial planting by reducing the population size.