TikTok, a social media platform for creating and sharing short videos, has seen a surge in popularity during the COVID-19 pandemic. To analyse the Italian vaccine conversation on TikTok, we downloaded a sample of videos with a high play count (Top Videos), identified through an unofficial Application Programming Interface (consistent with TikTok’s Terms of Service), and collected public videos from vaccine sceptic users through snowball sampling (Vaccine Sceptics’ videos). The videos were analysed using qualitative and quantitative methods, in terms of vaccine stance, tone of voice, topic, conformity with TikTok style, and other characteristics. The final datasets consisted of 754 Top Videos (by 510 single users) plus 180 Vaccine Sceptics’ videos (by 29 single users), posted between January 2020 and March 2021. In 40.5% of the Top Videos the stance was promotional, 33.9% were indefinite-ironic, 11.3% were neutral, 9.7% were discouraging, and 3.1% were ambiguous (i.e. expressing an ambivalent stance towards vaccines); 43% of promotional videos were from healthcare professionals. More than 95% of the Vaccine Sceptic videos were discouraging. Multiple correspondence analysis showed that, compared to other stances, promotional videos were more frequently created by healthcare professionals and by females, and their most frequent topic was herd immunity. Discouraging videos were associated with a polemical tone of voice and their topics were conspiracy and freedom of choice. Our analysis shows that Italian vaccine-sceptic users on TikTok are limited in number and vocality, and the large proportion of videos with an indefinite-ironic stance might imply that the incidence of affective polarisation could be lower on TikTok, compared to other social media, in the Italian context. Safety is the most frequent concern of users, and we recorded an interesting presence of healthcare professionals among the creators. TikTok should be considered as a medium for vaccine communication and for vaccine promotion campaigns.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12889-023-15748-y.