Silk fibroin (SF) is a natural polymeric biomaterial that is widely adopted for the preparation of drug delivery systems. Herein, we aimed to fabricate and characterize SF nanoparticles loaded with the selective estrogen receptor modulator; tamoxifen citrate (TC-SF-NPs) and to assess their in vitro efficacy against breast cancer cell lines (MCF-7 and MDA-MB-231). TC-loaded SF-NPs were characterized for particle size, morphology, entrapment efficiency, and release profile. In addition, we examined the in vitro cytotoxicity of TC-SF-NPs against human breast cancer cell lines and evaluated the anticancer potential of TC-SF-NPs through apoptosis assay and cell cycle analysis. Drug-loaded SF-NPs showed an average particle size of 186.1 ± 5.9 nm and entrapment efficiency of 79.08%. Scanning electron microscopy (SEM) showed the nanoparticles had a spherical morphology with smooth surface. Tamoxifen release from SF-NPs exhibited a biphasic release profile with an initial burst release within the first 6 h and sustained release for 48 h. TC-SF-NPs exerted a dose-dependent cytotoxic effect against breast cancer cell lines. In addition, flow cytometry analysis revealed that cells accumulate in G0/G1 phase, with a concomitant reduction of S- and G2-M-phase cells upon treatment with TC-SF-NPs. Consequently, the potent anticancer activities of TC-SF-NPs against breast cancer cells were mainly attributed to the induction of apoptosis and cell cycle arrest. Our results indicate that SF nanoparticles may represent an attractive nontoxic nanocarrier for the delivery of anticancer drugs.