Updated project metadata. Given the central role fertilization plays in the health and fit-ness of sexually reproducing organisms and the well-known evo-lutionary consequences of sexual selection and sperm compe-tition, knowledge gained by a deeper understanding of sperm(and associated reproductive tissues) proteomes has proven crit-5ical to the field’s advancement. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advance-ments in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophilasperm proteome was published. Here we provide an updated version of the Drosophila melanogastersperm proteome (DmSP3) using improved separation and detection methods andan updated genome annotation. We identified 2563 proteins,with label-free quantitation (LFQ) for 2125 proteins. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins. The top 20 most abundant pro-teins contained the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases (S-Laps). Both gene content and protein abundance were significantly reduced on the X chromosome, a finding consistent with prior genomic studies of the X chromosome gene content and evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. LFQ measured significant levels for 75/83 ribosomal proteins (RPs) we identified, including a number of core constituents. The role of this unique subset of RPs in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins found predominantly in the accessory glands. The possibility of tissue contamination from seminal vesicle or other reproductive tissues was addressed using concentrated salt and detergent treatments. Salt treatment had little effect on sperm proteome composition suggesting only minor contamination during sperm isolation while a significant fraction of Sfps remained associate dwith sperm following detergent treatment suggesting Sfps may arise within, and have additional functions in sperm per se.