While long-term plastic film mulching (LFM) of farmland can improve the yield and quality of crops, it also poses ecological risks through the accumulation of microplastics (MPs) in soil and alterations in soil nitrogen (N) cycling. However, no systematic studies (based on long-term experiments) have studied the effects of LFM on both soil MPs accumulation and gross N transformations. In this study, topsoils (0-20 cm) were collected from four LFM farmlands in Xinjiang, Liaoning, Sichuan, and Shandong provinces of China. The 15N isotope pool dilution method and Ntracebasic model were applied to quantify the impact of LFM on soil gross N transformation rates. Our results showed that LFM significantly increased the accumulation of MPs, particularly in the 0-10 cm layer. The gross N transformation rates varied among sites, reflecting regional differences in soil type. Mineralization rates increased in Xinjiang, Liaoning, and Sichuan under LFM, while microbial assimilation and autotrophic nitrification decreased in Xinjiang. In Sichuan, reduced soil nitrification potential led to low levels of mineral N (NH4+-N and NO3--N) retention. Furthermore, in Shandong, LFM decreased the mineralization potential of recalcitrant organic N but significantly enhanced heterotrophic nitrification. To improve predictions of agroecosystem N cycling, we show with this study that it is important to consider soil differences which drive gross N transformation rates associated with LFM.