To determine employee autonomous motivation and find out the effect of age and Gender on autonomous motivation among employees working in public sector medical university, Karachi. A Cross-sectional study design was used to conduct this study among employees of public sector medical university. The duration of the study was three months. The sample size of the study was 384. The response rate was 78%, 300 out of 384 duly filled questionnaires were collected. A probability random sampling technique was used to collect data. SPSS software was applied to analyze the collected data. Mean ±SD and % & n were explored for Quantitative and Qualitative variables, respectively. T-Test and one-way ANOVA were applied to test hypotheses. P-Value <0.05 was considered significant. 59.3% (n=178) were males. 46% (n=138) were females in age between 25 to 31 years. 53.7 % (n=161) respondent reported their marital status as single. The mean age of the respondents was 30.16±7.03. Factors that motivate employees included salary (34%), positive feedback (26.3%), felt very proud when did well at work (52%), learnt new things (57.7%), , had a feeling of accomplishment (35.3%), Solving work issues made them feel important (26.7%), felt guilty if didn’t accomplish a job-related task (45.3%). The t-test and ANOVA revealed significant effect of gender; t (298) = -2.81, p-value = 0.005, CI [-3.77, -0.66] and age; F (5,294) =2.56, p-Value = 0.027 on employees’ autonomous motivation. It was observed proven that there was a significant effect of gender and age on employee’s autonomous motivation.