Objective To investigate the effect of Shugan-Yishen formula on Tamoxifen-resistant MCF-7 cell line(LCC9) through HER2-ERK/MAPK-ERα pathway. Methods Four serums were prepared with the method of serum pharmacology:the serum of Shugan-Yishen formula, Shugan-Yishen formula plus Tamoxifen(TAM), blank and TAM alone. After LCC-9 cells were affected in the above-mentioed serums in vitro, the inhibition rate and the level of mRNA as well as the protein expression of HER2, ERα, ERK/MAPK, p-ERK/MAPK were detected. Results MTT showed that TAM alone group、Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had higher inhibition rates at 48th hour, the inhibition rates was(6.61±0.129)%, (47.43±2.24)%, (54.19±3.364)%, compared to the blank group, Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had a stronger inhibition on LCC9 at 48th hour (P<0.05), and the combination group was better than Shugan-Yishen formula alone(P<0.05), there was no difference between TAM group compared to the blank group(P>0.05). RT-PCR result showed that compared to the blank group, the expression levels of ESR1 mRNA of group of Shugan Yishen formula plus TAM werehigher by and large at 48th hour (P<0.05), the expression of ESR1 mRNA was(2.282±0.185);and the levels of ERBB2, MAPK3 mRNAwere lower(P<0.05), which levels were(0.71±0.095), (0.336±0.058). Western blotting result showed that Shugan-Yishen Formula plus TAM group could markedly decrease the expression of HER2, p-ERK/MAPK of LCC9 at 48th hour (P<0.05), which the expressions were(0.708± 0.072), (0.228±0.034), and the expression of ER-αelevated(P<0.05). There was no significant difference on ERK/MAPK(P>0.05). Conclusions ①The combination of Shugan-Yishen formula with tamoxifen can inhibit the LCC9 cells. The inhibition effect reaches the peak at 48th hour. ②The mechanism of Shugan-Yishen formula plus TAM combination effects on human breast cancer TAM-resistant cell line LCC9 may improve the sensitivity of ERαby inhibiting the expression of HER2, ERK/MAPK.