The purpose of this study was to analyze the autophagy of the human uterine myometrium during the labor. We collected uterine myometrium strips from term, singleton, nulliparous healthy women undergoing cesarean delivery before labor (nonlabor group, n = 10) or during normal labor (in-labor group, n = 10) without rupturing of membrane. The indications for cesarean delivery were breech presentation or maternal request. Transmission electron microscopy was used to observe autophagosomes. Reverse transcriptase polymerase chain reaction, immunofluorescence, and Western blot were used to quantify the messenger RNA (mRNA) and protein level of the autophagy markers LC3B, P62, and Beclin-1 in the uterine muscle strips. There were no differences between both groups in maternal age, body mass index, gestational week, neonatal weight, operative bleeding, and postpartum bleeding. Transmission electron micrographs showed that autophagosomes existed in myometrial tissue in both groups. There were more autophagosomes in the in-labor group than in the nonlabor group, and the difference had significance. The in-labor group had significantly greater LC3B mRNA expression but significantly lower P62 mRNA expression compared with the nonlabor group. Semiquantitative immunofluorescence in uterine myometrial cells in the in-labor group showed increased LC3B puncta formation and greater Beclin-1 expression but reduced P62 puncta formation compared with the nonlabor group. The ratio of LC3BII/I proteins was significantly higher, but P62 protein was significantly lower in the in-labor group compared with the nonlabor group. The Beclin-1 mRNA and protein expressions were not significantly different between the 2 groups. Autophagy was activated in human uterine myometrium during labor and might play an important role in maintaining uterine contraction function.