The habit of chewing tender leaves and twigs of khat is deep-rooted in Yemen. Khat cultivation causes several economical, environmental, social and medical problems in Yemen. This habit also has negative effects on the teeth and periodontal tissues, accordingly; the aim of this study was to evaluate the clinical, histological and immunohistochemical effects of khat chewing on the periodontium. Materials and Methods: 40 patients (both males and females) was classified to three groups, (Group I) thirty biopsies taken from the gingiva of the chewers only (15 chewing side and 15 from non-chewing side). (Group II) Thirty biopsies taken from the gingiva of khat chewers and smokers (15 chewing side and 15 from non-chewing side), and Group III included ten biopsies taken from the gingiva of the non-chewers and non-smokers. The biopsies were processed for histological study and according to manufacturer recommendations for staining with TNF-α and p53. Clinical examinations were performed including probing pocket depth, plaque index, gingival index and gingival recession. Results: The results showed that clinical parameters in group I did not differ significantly whereas in group II they showed that the chewing sides demonstrated higher PPD and CAL. On the other hand, GR in chewer sides of both group I and II showed statistically significantly higher mean values than non-chewer sides. P53 expression (area % and optical density) in the chewer sides was highly significant than non-chewer sides in both groups. In comparison of p53 area % between the three groups; P53 expression showed no statistically significant difference between Group I and Group II while Group III showed the statistically significantly lowest mean values. As for the optical density of P53 expression in the epithelium; Group II showed the statistically significant highest mean followed by Group I. Group III showed the significantly lowest mean. In the connective tissue, Group I showed the significantly highest mean followed by Group II then Group III showed the statistically significant lowest mean expression of p53. As for TNF-α expression, the area % in chewer sides showed statistically significant higher values than non- chewer sides in the connective tissue of group II and in the epithelium and connective tissues of group I. In comparison between the three groups, group II showed the statistically significant highest mean of TNF-α area% and optical density in both epithelium and connective tissue. This was followed by group I then group III. Conclusion: Khat chewing mainly caused clinical gingival recession on chewing sides with several histological changes that were more marked with associated smoking. Khat chewing caused over expression of p53 and TNF-α on chewing side and this aligns with the histological findings.