Conspecific males often disturb copulating mites, thus interrupting the mating and a subsequent mating may occur. However, in natural populations of Tetranychus urticae, virgin or sperm-depleted females exist (3.2% of the females in a population) and a second mating may occur when these females are placed with males of unknown age. How does multiple mating benefit the fitness of T. urticae females? To answer this question and to establish the success of re-mating, we investigated the effects of mating interruption at various time intervals after the start on the validity of fertilization. Female offspring ratios were significantly lower if mating was interrupted at 30, 60 or 90 s, compared with complete (undisturbed) mating. Females accepted the second mating if they could not obtain sufficient sperm from the first mating owing to interruption. The success of the second mating and priority of the second male's sperm depended on how soon the interruption of the first mating occurred. Proportion of females produced female offspring of the second males when mated at 6 days after the first mating was fewer than those when mated just after the first mating. To know the effect of males of unknown age, of which some were assumed to be sperm-depleted, we used them as the first mating partner and then used virgin males as the second mating partner. Three females (6.4%, n = 47) who mated with males of unknown age at the first mating, produced female offspring of the second males, but no females produced female offspring of the second males when mated with virgin males at the first mating. These findings indicate that sperm depletion, caused by mating interruption or paternal age in the first mating, might indeed affect the fertilizing ability.