Cigarette smoking is a leading cause of mortality worldwide. Various substances in cigarette smoke (CS) may either directly or indirectly, through inflammation, increase muscle proteolysis, inhibit protein synthesis, impair mitochondrial function and thereby cause skeletal muscle dysfunction. The long-term effects of CS on skeletal muscle have been widely explored, but to what extent these changes are reversible by smoking cessation is less known. To investigate this, we assessed the impact of smoking with or without 1 or 2 weeks of CS cessation on skeletal muscle structure and function in a mouse model. We hypothesized that short-term CS cessation reverses smoking-induced deterioration in muscle structure and function. Fifty-two C57Bl/6J mice were divided into 4 groups: chronic CS-exposure (for 14 weeks), 1 week cessation (13 weeks of CS and 1 week cessation), 2 weeks cessation (12 week CS and 2 weeks cessation) and controls (air-exposed for 14 weeks). CS-exposed mice were exposed via a nose-only system to mainstream CS of six 3R4F research cigarettes, twice daily. Comparison between groups was performed using ANOVA or Kruskal-Wallis tests. Animals were sacrificed with an intraperitoneal injection of an overdose of anesthesia. CS caused neutrophilic lung inflammation (p < 0.0001), reduced body (p < 0.001) and hindlimb muscle mass (p < 0.001), that returned to baseline after 1 (p < 0.05) to 2 weeks (p < 0.001) of smoking cessation. Dual-energy X-ray absorptiometry revealed reductions in fat mass (-26%, p = 0.0007) and had not returned to normal after 2 weeks of smoking cessation. Mitochondrial respiration in permeabilized soleus was assessed by high-resolution respirometry. The maximal uncoupled respiration and succinate/rotenone-stimulated respiration were lower in the CS-exposed group (p < 0.01) and similar to controls after 2 weeks of smoking cessation (p < 0.01), and total soleus protein levels of mitochondrial complexes (I, III and IV) showed a similar pattern (P < 0.05). Exposure of the soleus to CS extract also resulted in reduced respiration that was readily reversed after removal of the extract, indicating that substances in CS impair the function of respiratory chain complexes. Fiber sizes and capillarization were measured in the soleus, plantaris and diaphragm. While there was no effect in the soleus and plantaris, smoking reduced the sizes of all fiber types in the diaphragm (P < 0.001) and without capillary loss, this resulted in an increased capillary density (p < 0.01). Two weeks of smoking cessation restored these fiber sizes to normal and normalized capillary density while significantly reducing the heterogeneity of capillary spacing (p < 0.05) that is expected to improve muscle oxygenation. In conclusion, short-term smoking cessation reverses smoking-induced decrements in mitochondrial function and diaphragm muscle atrophy, highlighting immediate beneficial effects of smoking cessation.