The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure–function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full‐length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef‐mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21‐activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.