<p>Siphophages possess a long, flexible, and non-contractile tail that is responsible for host recognition, cell wall perforation, and genome delivery. Although the majority of structurally characterized siphophages target Gram-negative bacteria, those infecting Gram-positive bacteria remain elusive. Moreover, structural information concerning phage infection and genome release events in Gram-positive infecting siphophages is sparse. Here, we present a near-atomic resolution structure of <i>Brochothrix thermosphacta</i> bacteriophage NF5 determined by cryo-electron microscopy (cryo-EM). The structure comprises 11 proteins associated with the head, neck, tail tube, and baseplate, amounting to 643 polypeptides in total. Integration of cellular cryo-electron tomography (cryo-ET) showed the infection process of NF5, providing insights into the adsorption mechanism of siphophages targeting Gram-positive bacteria. Structural comparisons of baseplates from multiple siphophages targeting Gram-negative and Gram-positive bacteria reveal divergent compositional architectures and distinct assembly mechanisms. These disparities are evident in the domains across divergent baseplate proteins, suggesting evolutionary adaptations to host envelope architectures.</p>