Osteoclasts are giant bone-digesting cells that harbour specialized organelles termed ‘secretory lysosomes’ that give rise to the ruffled border, the osteoclasts’ bone-resorbing apparatus. Fusion of secretory lysosomes with the ruffled border equips its membrane folds with molecular machinery required to dissolve bone mineral and degrade the underlying organic matrix. Understanding the nature and contribution of secretory lysosome proteins therefore holds potential to discover new regulators of osteoclast function and bone homeostasis. Using a proteomic approach, we recently resolved the molecular landscape of osteoclast secretory lysosomes (Ng, Ribet et al., Nat Commun, 2023). Here to further validate the osteoclast secretory lysosome proteome, we interrogated the functional contribution(s) of several proteins predicted to participate in secretory lysosome membrane transport or trafficking in osteoclasts using an RNAi-based screen. High-confidence secretory lysosome membrane proteins (LogFC > 1.5 and p<0.05) were specifically depleted in osteoclasts using gene targeted siRNAs. Knockdown efficiency was monitored by assessing the mRNA and protein levels by quantitative PCR and immunoblotting, respectively. Optical, reflective and high-resolution confocal microscopy was used to assess the impact of targeted gene suppression on key osteoclast parameters including: (i) differentiation; (ii) polarization (iii) extracellular acidification; (iv) bone resorption on devitalized bone discs and; (v) endo-lysosomal morphology by immunofluorescence and electron microscopy. Remarkably, despite all being constituents of secretory lysosome membranes, knockdown of individual target genes resulted in differential and unexpected changes in (i) osteoclast morphology, (ii) polarisation and (iii) their capacity to acidify and digest bone. In addition, knockdown of several genes elicited striking morphological disturbances in the size and positioning of osteoclast endo-lysosomes. Together, this work unmasks several new regulators of osteoclast formation, bone resorption and endo-lysosomal homeostasis, highlighting the value of coupling ‘omic’ based datasets with in vitro studies in physiologically relevant cells to validate potential regulators of osteoclast function.