Primary uterine inertia (PUI) is the most common type of dystocia in dogs. We hypothesized that PUI develops because of lower than normal expression of the basic contractile elements in the uterus, i.e., smooth muscle (SM) α- and γ-actin and SM-myosin, and that the expression of these proteins is influenced by the number of fetuses present in utero. Full-thickness inter-placental uterine biopsies were collected during Cesarean sections from dogs with PUI (n = 11), and from bitches with obstructive dystocia (OD) still presenting strong labor contractions (designated as the control group, n = 7). Relative gene expression was determined by semi-quantitative real-time (TaqMan) PCR, and protein localization by immunohistochemistry. Gene expression between PUI and OD bitches, and between PUI bitches carrying small, large, or average number of fetuses according to their breed, were compared. Uterine SM-γ-actin and SM-myosin mRNA levels were significantly higher in PUI than in OD dogs, while SM-α-actin did not differ. PUI bitches carrying large litters had lower uterine SM-γ-actin gene expression than those with small litters (P = 0.008). Immunostaining for SM-actin isoforms and SM-myosin was present in the myometrium, and localization pattern and staining intensity appeared similar in the PUI and OD groups. All proteins stained in blood vessels, and SM-γ-actin was also present in endometrial luminal and glandular epithelium. In conclusion, higher uterine SM-γ-actin and SM-myosin gene expression in PUI bitches, compared with OD dogs, might be an indication of abnormal progression with labor. Whether this is the cause of PUI due to an intrinsic error of the myometrium not becoming committed to labor, or the consequence of inadequate endocrine or mechanical stimuli, is not clear. Litter size was previously shown to be one of the risk factors for the development of uterine inertia in dogs, and our findings suggest possible differing uterine pathophysiology of PUI with respect to litter size.