In preparation for fertilization, mammalian oocytes undergo optimization of the mechanisms that regulate calcium homeostasis. Among these changes is the increase in the content of the Ca(2+) stores ([Ca(2+)]ER), a process that requires Ca(2+) influx. Nevertheless, the mechanism(s) that mediates this influx remains obscure, although is known that [Ca(2+)]ER can regulate Ca(2+) influx via store-operated Ca(2+) entry (SOCE). We find that during maturation, as [Ca(2+)]ER increases, Ca(2+) influx decreases. We demonstrate that mouse oocytes/eggs express the two molecular components of SOCE--stromal interaction molecule 1 (Stim1) and Orai1--and expression of human (h) Stim1 increases Ca(2+) influx in a manner that recapitulates endogenous SOCE. We observe that the cellular distribution of hStim1 and hOrai1 during maturation undergoes sweeping changes that curtail their colocalization during the later stages of maturation. Coexpression of hStim1 and hOrai1 enhances influx throughout maturation but increases basal Ca(2+) levels only in GV oocytes. Further, expression of a constitutive active form of hStim1 plus Orai1, which increases basal Ca(2+) throughout maturation, disturbs resumption of meiosis. Taken together, our results demonstrate that Ca(2+) influx and SOCE are regulated during maturation and that alteration of Ca(2+) homeostasis undermines maturation in mouse oocytes.