Our previous study demonstrated that deficiency of CD38‐NAADP signaling led to macrophage lysosomal free cholesterol accumulation and atherosclerosis in CD38−/− mice. The present study was designed to explore the underlining mechanism of lysosomal free cholesterol buildup in atherogenesis. By fluorescence microscopic analysis, we found that lysosomal Ca2+ release responses were significantly attenuated and lysosomal lumen acidity markedly decreased upon the inhibition of CD38‐NAADP signaling by silencing CD38, bafilomycin A1 (Baf, 100 μM), or NED‐19 (10 μM) in primary peritoneal CD38+/+ mouse macrophages on oxLDL (10 μg/ml). Consistently, reduced lysosomal Ca2+ release responses and declined lysosomal lumen acidity were also observed in CD38−/− macrophages treated with oxLDL. The secretion of IL‐1β from CD38+/+ macrophages was significantly increased with the inhibition of CD38/NAADP. Furthermore, the elevated plasma IL‐1β was observed in atherosclerotic CD38−/− mice on the Western diet. These results for the first time indicate that lysosomal Ca2+ depletion, lumen acidity decrease, and the increased secretion of proinflammatory factors may be the atherogenic mechanism of lysosomal free cholesterol buildup in the CD38‐NAADP signaling deficient macrophages (Supported by R01HL091464, NIH R01HL115068 and R01HL057244).