Modulation of autophagy has been considered as a potential therapeutic target for cancers and increasing efforts have been focused on autophagy inhibition in combination with chemotherapeutic agents. In this study, we identified that pulsatilla saponin D (PSD) is a potent lysosomal deacidification agent and its combination with camptothecin (CPT) synergistically inhibited the growth of both MCF-7 and MDA-MB-231 breast cancer cells. Co-treatment of PSD and CPT blocked autophagosome-lysosome fusion via altering the acidity of lysosome and downregulating lysosomal cathepsins, leading to an increased number of EGFP-LC3B punctate dots and high levels of LC3II and p62 in the two breast cancer cell lines. In addition, suppression of autophagy by PSD and CPT in combination triggered aggregation of ubiquitinated proteins through inducing p62 accumulation. P62 deletion almost completely normalized the increased levels of ubiquitinated proteins induced by the combination treatment. Notably, excessive expression of p62 significantly augmented these compounds combination-induced cytotoxicity in a time-dependent manner. Collectively, these data suggest that PSD could function as a novel autophagy inhibitor which could be used in combination with chemotherapeutic agents to exert synergistic anticancer effect in breast cancer treatment.