Checkpoint 300 serves a large amount of Palestinian labourers as they make their way to places of employment in East Jerusalem and Israel. The Checkpoint is a large complex of corridors, turnstiles, metal detectors and security desks that control the movement and mobility of these workers every day, subjecting them to enforced waiting, stress and absence from the home. In this article we examine the Checkpoint as a regulatory site of Israeli state biopolitics that, by distributing bodies and affects is productive of particular subjects and practices. We articulate our approach to biopolitics through a focus on bodies, discipline and affect before drawing on research visits to give an account of how the space of Checkpoint 300 enacts corporeal and affective discipline. We discuss the Checkpoint as a complex space that is functional, hierarchical and subjectivising and propose that the Checkpoint produces and governs a heteronormative sexual division of labour that is conducive to Israeli state biopolitics by a) upholding patriarchal relations and b) producing a docile male Palestinian labour force to build settlements for the Israeli population. We thus argue that the subject-making processes at Checkpoint 300 work to differentiate and govern Palestinian bodies in ways that are tied to the broader biopolitical objectives of the Israeli state. We close with reflections on the contributions of such an understanding of checkpoints in Palestine and draw attention to the important future lines of inquiry indicated by the research.