Simple SummaryEnvironment-friendly and sustainable insect pest control approaches such as the sterile insect technique/inherited sterility (SIT/IS) have been proposed against the European grapevine moth Lobesia botrana. Reproductive behaviour, such as oviposition, mating ability, and multiple mating of 150 Gy-treated females, was studied. Moreover, the last-male sperm precedence (P2 value) and male’s flight response to treated females were also investigated. The present study aims to provide basic information which is essential to ensure successful implementation of SIT/IS as part of an integrated approach for the management of L. botrana.The sterile insect technique/inherited sterility (SIT/IS) has been suggested as an eco-friendly control tactic for area-wide integrated pest management approaches in order to control the European grapevine moth, Lobesia botrana. This study assessed the effects of an irradiation dose of 150 Gy administered to newly emerged female moths on their egg laying behaviour and mating ability at different ages at mating. Moreover, the effects of multiple mating on the mating ability of treated females, pattern of sperm precedence in twice-mated females and the flight response of treated and untreated males to treated and untreated calling females were also investigated. Females treated with 150 Gy initiated calling in a way similar to untreated females. When treated females were paired with untreated males, the mean number of eggs oviposited per female during 6 days was reduced (59.6 and 82.8 eggs/female, respectively), as was their mating ability and multiple mating compared with untreated females. The proportion of offspring fertilized by the second of the two males to mate with the female or last-male sperm precedence (P2 value) constituted 97% of the eggs, suggesting that the second male mate fathered the most offspring. The outcome of this work could be viewed as an integrated approach for improving effectiveness and enabling successful implementation of a SIT/IS program against L. botrana.