The dysregulation of miRNAs has been increasingly recognized as a critical mediator of cancer development and progression. Here, we show that frequent deletion of theMIR135A1locus is associated with poor prognosis in primary breast cancer. Forced expression of miR-135a decreased breast cancer progression, while inhibition of miR-135a with a specific miRNA sponge elicited opposing effects, suggestive of a tumor suppressive role of miR-135a in breast cancer. Estrogen receptor alpha (ERα) bound the promoter ofMIR135A1for its transcriptional activation, whereas tamoxifen treatment inhibited expression of miR-135a in ERα+breast cancer cells. miR-135a directly targetedESR1, ESRRA,andNCOA1, forming a negative feedback loop to inhibit ERα signaling. This regulatory feedback between miR-135a and ERα demonstrated that miR-135a regulated the response to tamoxifen. The tamoxifen-mediated decrease in miR-135a expression increased the expression of miR-135a targets to reduce tamoxifen sensitivity. Consistently, miR-135a expression was downregulated in ERα+breast cancer cells with acquired tamoxifen resistance, while forced expression of miR-135a partially resensitized these cells to tamoxifen. Tamoxifen resistance mediated by the loss of miR-135a was shown to be partially dependent on the activation of the ERK1/2 and AKT pathways by miR-135a–targeted genes. Taken together, these results indicate that deletion of theMIR135A1locus and decreased miR-135a expression promote ERα+breast cancer progression and tamoxifen resistance.