Background: Studies have shown that the axis of the urokinase-type plasminogen activator (uPA–uPAR) has a pleiotropic effect in different stages of cancer. The purpose of the study was to reveal characteristics of the B16/F10 melanoma development in mice with the uPA gene knockout with and without chronic neurogenic pain (CNP). Methods: The study included male and female С57ВL/6 mice (n = 102) and C57BL/6-Plautm1.1BugThisPlauGFDhu/GFDhu mice with the uPA gene knockout (n = 48). B16/F10 melanoma was transplanted subcutaneously to the animals in the main groups 2 weeks after bilateral sciatic nerve ligation (CNP model). Mice with the standard melanoma transplantation were the controls. Results: The survival of uPA gene-knockout mice with melanoma was similar to that in controls and was 1.5 times (p < 0.05) longer in females than in males; the melanoma onset in these mice was observed a week earlier; the dynamics of tumor growth showed a pronounced gender dependence: in females, the tumor practically did not grow, and its volume before the death did not exceed 1.0 cm³, while in males the tumor was characterized by an active growth with two peaks of the volume increase (weeks 2 and 4); melanoma metastasized weakly - single metastases to the lungs (in females) or no metastases but with hemorrhages in the lungs and heart (in males). CNP reduced the survival of uPA gene-knockout females, similar to mice with the normal genome, but did not affect the survival of males; primary tumors in uPA gene-knockout mice emerged a few days later than in the control, but grew more intensively, with gender differences smoothed out; increased metastasis was demonstrated by the initiation of metastatic lesions of the lungs and liver in males, while hemorrhages in the lungs maintained, and by an increased number of metastases in the lungs with the appearance of lung hemorrhages in females. Conclusions: The uPA gene knockout differently changes the course of B16/F10 melanoma in male and female mice. CNP enhances the tumor growth, erasing gender differences, and activates metastasis of melanoma.