The present study explored whether calcitriol plays a role in the regulation of sodium-dependent glucose transporter protein 1 (SGLT1) activity. For this purpose, alpha-methyl glucoside (AMG) uptake in stable transfected Chinese hamster ovary (CHO-G6D3) cells expressing rabbit SGLT1 (rbSGLT1) was used. The involvement of second messengers, intracellular signaling pathways, and pro-inflammatory cytokines were examined using specific inhibitors before incubation with calcitriol for 15 min. The present study demonstrated the involvement of second messengers produced by phospholipase A(2), phospholipase C, calmodulin, diacylglycerol kinase, and phosphoinositide 3 kinase on calcitriol-regulated AMG uptake. Pretreatment with inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway increased calcitriol-induced AMG uptake. In contrast, inhibition of the phosphoinositide 3-kinase PI3K/Akt/mTOR signaling pathway decreased the effect of calcitriol on AMG uptake. These findings suggest that calcitriol regulates rbSGLT1 activity through a rapid, extranuclear initiated mechanism of action stimulated by MAPK and inhibited by PI3K/Akt/mTOR. Another important finding was the effect of pro-inflammatory cytokines on calcitriol-induced AMG uptake. Interleukin-6 increased while tumor necrosis factor-alpha decreased calcitriol-induced AMG uptake. In conclusion, the present study demonstrates the involvement of calcitriol in the regulation of rbSGLT1 activity. This is due to the activation of intracellular signaling pathways triggered by second messenger molecules and cytokines after a short time (15 min) exposure to calcitriol.