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Summary The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed., Highlights • VAMP4 is the first identified ADBE cargo • VAMP4 is essential for ADBE • Most synaptic vesicle cargoes are not selectively recovered by ADBE, Nicholson-Fish et al. show that VAMP4 is selectively retrieved by activity-dependent bulk endocytosis (ADBE) and is essential for ADBE to proceed. Thus, synaptic vesicles generated by ADBE will have a specific molecular composition that defines their physiological function.