In this paper, the tribological behavior of 316L stainless steel with heterogeneous lamella structure (HLS), prepared through 85% cold rolling technology and subsequent annealing treatment (750 °C, 10 min), were conducted on a ball-on-disc tribometer under different normal loads in dry ambient air conditions. The morphologies, structures, and compositions of the raw and worn surfaces were analyzed by 3D surface profilometer, XRD, SEM, EDS and TEM. Based on this, the results showed that the HLS 316L stainless steel samples exhibited lower and more steady friction coefficients than coarse-grained samples, especially under higher loads, which can be attributed to the existence of numerous oxidative particles across sliding interfaces. However, the wear resistance of HLS 316L stainless steel sample was a little weakened compared to that of the coarse-grained sample under a normal load of 5 N. When the load increases up to 15 N, an obviously decreased wear resistance was found for the HLS of the 316L stainless steel sample, which was 50% lower than that of coarse-grained sample. This can be ascribed to the more severe oxidative and abrasive wear performance of HLS 316L stainless steel sample under dry sliding conditions.