Previous reports have shown that estrogen, which changes in pregnancy levels, participates in GI motility regulation and is involved in the pathogenesis of various functional disorders in the stomach. The current study aimed to explore the changes in the expression of estrogen receptors (ERs) and examine the effect of estrogen on nitric oxide- (NO-) cyclic guanosine monophosphate (cGMP) pathway and thus relaxation in gastric smooth muscle cells (GSMC) during pregnancy. Researchers measured protein and mRNA expression levels of ERs via specifically designed enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), respectively. NO, and Researchers measured cGMP levels via specifically designed ELISA kits. Estrogen increased both NO and cGMP levels, and their levels were more significant in early compared to late pregnancy. Expression of ERs was more remarkable in early compared to late pregnancy. ACh induced a more significant contraction of GSMC in late pregnancy compared to early pregnancy. Estrogen inhibited ACh-induced contraction in both periods of pregnancy. Significantly, pretreatment of GSMC with either L-NNA or ODQ abolished estrogen inhibitory action on muscle contraction. In conclusion, GSMC contractile behavior undergoes drastic changes in response to estrogen during pregnancy, which might explain some of the pregnancy-associated gastric disorders.