PD-L1 is one of the most important immune checkpoint molecules in breast cancer that plays an important role in suppressing the immune system when confronted with tumor cells and is regulated by various microRNAs. Among them, microRNA-335-3p and microRNA-145-5p, regulated by DNA methylation, have tumor suppressor activities. We studied the role of miR-335 and -145 on PD-L1 suppression in breast cancer. The expression of miR-355 and miR-145 was significantly downregulated in BC tissues and cell lines compared to their controls, and their downregulation was negatively correlated with PD-L1 overexpression. In-silico and luciferase reporter systems confirmed that miR-335 and -145 target PD-L1. In BC tissues and cell lines, cancer-specific methylation was found in CpG-rich areas upstream of miR-335 and-145, and up-regulation of PD-L1 expression was connected with hypermethylation (r = 0.4089, P = 0.0147, and r = 0.3373, P = 0.0475, respectively). The higher levels of miR-355 and -145 in BC cells induced apoptosis, arrested the cell cycle, and reduced proliferation significantly. In summary, we found that miR-335 and -145 are novel tumor suppressors inactivated in BC, and these miRs may serve as potential therapeutic targets for breast cancer treatment.