The aim of this work was to determine the expression of the ERβ (estrogen receptor β) and multidrug resistance, namely MDR1 (P-glycoprotein, P-gp), in 152 samples of non-small cell lung cancer. The expression pattern of ERβ and MDR1 were assessed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry. We also analyzed the correlation between ERβ and MDR1 with clinical and pathological data. The co-expression pattern of ERβ and individual MDR1 proteins was assessed by correspondence analysis and chi-squared tests. In the present study, we found that patients with tumor stage I-II showed higher ERβ mRNA expression levels and decreased expression of ERβ protein with increasing tumor grade, which is opposite to MDR1 expression. In addition, an opposite co-expression pattern of ERβ and individual MDR1 proteins was also observed. In conclusion, the results can be used to better understand the expression control of MDR1 and may allow for the establishment of new cancer chemistry strategies that will control P-gp expression in NSCLC.