To explore the relationship between signal-stimulated increases in intracellular calcium ([Ca² ]_i) and depletion and refilling of the endoplasmic reticulum (ER) Ca² stores ([Ca² ]_L) in human myometrial cells, we measured simultaneous changes in [Ca² ]_i and [Ca² ]_L using Fura-2 and Mag-fluo-4, respectively, in PHM1-41 immortalized and primary cells derived from pregnant myometrium and in primary cells derived from nonpregnant tissue. Signal- and extracellular Ca² -dependent increases in [Ca² ]_i (SRCE) and ER refilling stimulated by oxytocin and cyclopiazonic acid were not inhibited by voltage-operated channel blocker nifedipine or mibefradil, inhibition of Na /Ca² exchange with KB-R7943, or zero extracellular Na in PHM1-41 cells. Gadolinium-inhibited oxytocin- and cyclopiazonic acid-induced SRCE and slowed ER store refilling. TRPC1 mRNA knockdown specifically inhibited oxytocin-stimulated SRCE but had no statistically significant effect on ER store refilling and no effect on either parameter following cyclopiazonic acid treatment. Dominant negative STIMΔERM expression attenuated oxytocin- and thapsigargin-stimulated SRCE. Both STIM1 and ORAI1–ORAI3 mRNA knockdowns significantly attenuated oxytocin- and cyclopiazonic acid-stimulated SRCE. The data also suggest that reduction in STIM1 or ORAI1–ORAI3 mRNA can impede the rate of ER store refilling following removal of SERCA inhibition. These data provide evidence for both distinct and overlapping influences of TRPC1, STIM1, and ORAI1–ORAI3 on SRCE and ER store refilling in human myometrial cells that may contribute to the regulation of myometrial Ca² dynamics. These findings have important implications for understanding the control of myometrial Ca² dynamics in relation to myometrial contractile function.